Purified amniotic membrane compositions and methods of use

ABSTRACT

Compositions having a combination of specific biological components have been found to exert a number of useful effects in mammalian cells, including modulating TGF β signaling, apoptosis, and proliferation of mammalian cells, as well as decreasing inflammation in mice. These components can be obtained commercially, or can be prepared from biological tissues such as placental tissues. Placental amniotic membrane (AM) preparations described herein include AM pieces, AM extracts, AM jelly, AM stroma, and mixtures of these compositions with additional components. The compositions can be used to treat various diseases, such as wound healing, inflammation and angiogenesis-related diseases.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/720,760, filed Sep. 27, 2005, which is incorporated herein byreference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The invention was made with United States government support under grantnumber RO1 EY06819 awarded by the National Institutes of Health. TheUnited States government may have certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the fields of biology andpharmaceuticals. More particularly, the invention relates tocompositions and methods for modulating cellular physiology andpathological processing using a combination of compounds that can befound in amniotic membrane preparations.

BACKGROUND OF THE INVENTION

The placenta is a temporary organ that surrounds the fetus duringgestation. The placenta allows for transport of gases and nutrients, andalso provides other metabolic and endocrine functions. The placenta iscomposed of several tissue types. The umbilical cord connects theplacenta to the fetus, and transports oxygen to the fetus. The umbilicalcord has two arteries and a vein. Wharton's jelly, a specializedgelatinous connective tissue material, surrounds the umbilical cord toprotect it from damage during fetal movement and development. The outer“shell” of the placenta is known as the “chorion.” Much of the placentaldisc is composed of chorionic villi, which are extensions of thechorionic villous tree. Through these structures, fetal nutritionexchange occurs. The amniotic membrane (AM) is an avascular membranoussac that is filled with amniotic fluid. This membrane is the innermostmembrane surrounding a fetus in the amniotic cavity. This tissueconsists of an epithelial layer and a subadjacent avascular stromallayer.

SUMMARY OF THE INVENTION

Described herein are purified compositions and amniotic membranepreparations (that is, compositions that are prepared from amnioticmembrane materials, including the amniotic membrane, amniotic stroma andamniotic jelly). In some embodiments, at least one component of thepurified compositions are obtained from amniotic membrane preparations.Also described herein are purified compositions in which at least onecomponent of the purified composition is obtained from human placentaand chorion. Also described herein are methods for preparing any of theaforementioned purified compositions and preparations. Also describedherein are methods for storing and preserving any of the aforementionedpurified compositions and preparations. Also described herein aremethods for using any of the aforementioned purified compositions andpreparations, including preservative methods, cell culture methods,tissue culture methods, therapeutic methods, prophylactic methods andcosmetic methods.

Various AM preparations exert a number of physiologically significanteffects in mammalian cells and intact mammalian tissues. Such effectsinclude suppressing TGF β signaling, increasing apoptosis ofmacrophages, decreasing cellular proliferation of, decreasing cellularmigration of, and increasing apoptosis of vascular endothelial cells,protecting corneal and limbal epithelial cells and keratocytes fromapoptosis induced by storage or by dispase treatment, and decreasinginflammation in tissues. In addition to pieces of intact AM, otherpreparations described herein include pieces of AM stroma, processed(e.g., ground or pulverized) AM or AM stroma, and various extracts ofintact AM and AM stroma. AM extracts can be in liquid or lyophilizedpowder form.

The compositions also include thickened or gel forms of AM extractswhich can be made by mixing the AM extracts with a thickener such as oneor more extra cellular matrix components (ECM). A large number of ECMcomponents are known such as collagen, hyaluronic acid (HA), and fibrin.In some aspects, a purified composition having cross-linked highmolecular weight hyaluronan (HA), Tumor necrosis factor-stimulated gene6 (TSG-6), Pentraxin (PTX-3),and Thrombospondin (TSP-1) is provided. Atleast a portion of the components can be prepared from human amnioticmaterial such as human amniotic membrane, a human amniotic jelly, ahuman amniotic stroma, or a combination thereof. The composition canalso contain Smad7. The cross-linking of the HA can be, for example, acovalent bond to a heavy chain of inter-α-trypsin inhibitor. The ratioof protein to HA can be less than about 100. The composition can beprepared by obtaining a frozen or previously-frozen human placenta,thawing the placenta and isolating the human amniotic material from thethawed placenta, and homogenizing the human amniotic material in asuitable buffer. The preparation can also include, for example, freezingthe human amniotic material and griding the frozen amniotic material.The preparation can also include, for example, lyophilizing thehomogenate, or centrifuging the homogenate and isolating the supernatantfrom the centrifuged homogenate. The preparation procedure can alsoinclude, for example, lyophilizing the supernatant into powder. Thecomposition can also have a pharmaceutically acceptable carrier for anon-solid dosage form or an extended release solid dosage form.

Although preparations, materials, and methods similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, suitable preparations, methods and materials aredescribed herein. All publications mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions will control. In addition, theparticular embodiments discussed below are illustrative only and notintended to be limiting.

Certain Definitions

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

“Antioxidants” include, for example, butylated hydroxytoluene (BHT),sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. Incertain embodiments, antioxidants enhance chemical stability whererequired.

“Binders” impart cohesive qualities and include, e.g., alginic acid andsalts thereof; cellulose derivatives such as carboxymethylcellulose,methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®),ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g.,Avicel®); microcrystalline dextrose; amylose; magnesium aluminumsilicate; polysaccharide acids; bentonites; gelatin;polyvinylpyrrolidone/vinyl acetate copolymer; crosspovidone; povidone;starch; pregelatinized starch; tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), and lactose; a natural or synthetic gum suchas acacia, tragacanth, ghatti gum, mucilage of isapol husks,polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone®XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodiumalginate, and the like.

The term “carrier,” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues.

“Carrier materials” include any commonly used excipients inpharmaceutics and should be selected on the basis of compatibility withcompounds disclosed herein, and the release profile properties of thedesired dosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. “Pharmaceutically compatible carrier materials” may include, butare not limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The term “delayed release” as used herein refers to the delivery so thatthe release can be accomplished at some generally predictable locationmore distal to that which would have been accomplished if there had beenno delayed release alterations.

“Dispersing agents,” and/or “viscosity modulating agents” includematerials that control the diffusion and homogeneity of a drug throughliquid media or a granulation method or blend method. In someembodiments, these agents also facilitate the effectiveness of a coatingor eroding matrix. Exemplary diffusion facilitators/dispersing agentsinclude, e.g., hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolcan have a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizcers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents particularly useful in liposomal dispersions and self-emulsifyingdispersions are dimyristoyl phosphatidyl choline, natural phosphatidylcholine from eggs, natural phosphatidyl glycerol from eggs, cholesteroland isopropyl myristate.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution. In certain embodiments,diluents increase bulk of the composition to facilitate compression orcreate sufficient bulk for homogenous blend for capsule filling. Suchcompounds include e.g., lactose, starch, mannitol, sorbitol, dextrose,microcrystalline cellulose such as Avicel®; dibasic calcium phosphate,dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate;anhydrous lactose, spray-dried lactose; pregelatinized starch,compressible sugar, such as Di-Pac® (Amstar); mannitol,hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetatestearate, sucrose-based diluents, confectioner's sugar; monobasiccalcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactatetrihydrate, dextrates; hydrolyzed cereal solids, amylose; powderedcellulose, calcium carbonate; glycine, kaolin; mannitol, sodiumchloride; inositol, bentonite, and the like.

An “enteric coating” is a substance that remains substantially intact inthe stomach but dissolves and releases the drug in the small intestineor colon. Generally, the enteric coating comprises a polymeric materialthat prevents release in the low pH environment of the stomach but thationizes at a higher pH, typically a pH of 6 to 7, and thus dissolvessufficiently in the small intestine or colon to release the active agenttherein.

“Filling agents” include compounds such as lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol,mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition including a compound as disclosed herein required to providea clinically significant decrease in disease symptoms without undueadverse side effects. An appropriate “effective amount” in anyindividual case may be determined using techniques, such as a doseescalation study. The term “therapeutically effective amount” includes,for example, a prophylactically effective amount. An “effective amount”of a compound disclosed herein, is an amount effective to achieve adesired pharmacologic effect or therapeutic improvement without undueadverse side effects. It is understood that “an effect amount” or “atherapeutically effective amount” can vary from subject to subject, dueto variation in metabolism of the composition, age, weight, generalcondition of the subject, the condition being treated, the severity ofthe condition being treated, and the judgment of the prescribingphysician.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

The terms “kit” and “article of manufacture” are used as synonyms.

The term “modulate,” as used herein, means to interact with a targeteither directly or indirectly so as to alter the activity of the target,including, by way of example only, to enhance the activity of thetarget, to inhibit the activity of the target, to limit the activity ofthe target, or to extend the activity of the target.

As used herein, the term “modulator” refers to a compound that alters anactivity of a molecule. For example, a modulator can cause an increaseor decrease in the magnitude of a certain activity of a moleculecompared to the magnitude of the activity in the absence of themodulator. In certain embodiments, a modulator is an inhibitor, whichdecreases the magnitude of one or more activities of a molecule. Incertain embodiments, an inhibitor completely prevents one or moreactivities of a molecule. In certain embodiments, a modulator is anactivator, which increases the magnitude of at least one activity of amolecule. In certain embodiments the presence of a modulator results inan activity that does not occur in the absence of the modulator.

The term “non water-soluble diluent” represents compounds typically usedin the formulation of pharmaceuticals, such as calcium phosphate,calcium sulfate, starches, modified starches and microcrystallinecellulose, and microcellulose (e.g., having a density of about 0.45g/cm3, e.g. Avicel, powdered cellulose), and talc.

By “pharmaceutically acceptable,” as used herein, refers to a material,such as a carrier or diluent, which does not abrogate the biologicalactivity or properties of the compound, and is relatively nontoxic,i.e., the material may be administered to an individual without causingundesirable biological effects or interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. the AM preparations and purified compositionsdescribed herein and a co-agent, are both administered to a patientsimultaneously in the form of a single entity or dosage. The term“non-fixed combination” means that the active ingredients, e.g. the AMpreparations and purified compositions described herein and a co-agent,are administered to a patient as separate entities eithersimultaneously, concurrently or sequentially with no specificintervening time limits, wherein such administration provides effectivelevels of the two compounds in the body of the patient. The latter alsoapplies to cocktail therapy, e.g. the administration of three or moreactive ingredients.

“Plasticizers” are compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. In someembodiments, plasticizers can also function as dispersing agents orwetting agents.

The term “polypeptide” or “protein” as used herein can be the fulllength polypeptide, or a fragment or segment of a polypeptide, and canencompass a stretch of amino acid residues of at least about 8 aminoacids, generally at least 10 amino acids, more generally at least 20amino acids, often at least 30 amino acids, more often at least 50 aminoacids or more of the full length polypeptide.

“Solubilizers” include compounds such as triacetin, triethylcitrate,ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

“Stabilizers” include compounds such as any antioxidation agents,buffers, acids, preservatives and the like.

“Substantially pure” or “purified” when used in the context of abiological material, amniotic materia and/or a protein context typicallymeans that the material is isolated from other contaminating proteins,nucleic acids, and other biologicals derived from the original sourceorganism. Purity, or “isolation” may be assayed by standard methods, andwill ordinarily be at least about 10% pure, more ordinarily at leastabout 20% pure, generally at least about 30% pure, and more generally atleast about 40% pure; in further embodiments at least about 50% pure, ormore often at least about 60% pure; in still other embodiments, at leastabout 95% pure.

“Suspending agents” include compounds such as polyvinylpyrrolidone,e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g.,the polyethylene glycol can have a molecular weight of about 300 toabout 6000, or about 3350 to about 4000, or about 7000 to about 5400,sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

“Surfactants” include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Someother surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. In some embodiments, surfactants may be included toenhance physical stability or for other purposes.

As used herein, the term “subject” is used to mean an animal, preferablya mammal, including a human or non-human. The terms patient and subjectmay be used interchangeably.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating a disease or condition symptoms,preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

“Wetting agents” include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS , ammonium saltsand the like.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A is a non-limiting example of a bar graph showing the suppressionof TGF-β1 promoter activity by various AM Extracts. PL: plastic control.FRO/P: frozen amniotic membrane, placental portion. FRO/F: frozenamniotic membrane, fetal portion. FRE/P: Fresh amniotic membrane,placental portion. FRE/F: Fresh amniotic membrane, fetal portion. FIG.1B is a table comparing the P values of the various placentalpreparations.

FIG. 2 is a non-limiting example of a bar graph showing the doseresponse curve of TGF-β1 promoter activity suppression. RLU: Relativeluciferase units.

FIG. 3 is a non-limiting example of a bar graph showing the effect ofvarious AM extract preparations on the suppression of TGF-βRII promoteractivity.

FIG. 4 is a non-limiting example of a bar graph showing the effect ofvarious AM extract preparations on the suppression of TGF-βRII promoteractivity.

FIG. 5 is a non-limiting example of a bar graph demonstrating thatsoluble AME and jelly extracts derived after centrifugation do not alterthe suppressive effect on TGF-β Promoter Activities. HA, AM (Total (T),Low Speed (LS), High speed (HS)) and Jelly (Total (T), Low Speed (LS),High Speed (HS)) showed suppression of TGF-β□1 promoter activationcompared to the PBS control when normalized with beta-galatosidaseactivity.

FIG. 6 is a non-limiting example of a set of microscopic images of humancorneal fibroblasts showing cell morphology changes either 18 or 48hours after treatment with various compounds. PBS: the PBS control; HA:hyaluronic acid; AME: amniotic membrane extract; L/AME: lyophilizedamniotic membrane extract; AMJ: amniotic membrane jelly; L/AMJ:lyophilized amniotic membrane jelly.

FIG. 7 is a non-limiting example of a bar graph demonstrating the effectof AME (at 25 or 125 μg/ml), with or without lyophilized (L), on thesuppression of TGFβ1 activity. The activity is measured in relativeluciferase units (RLU).

FIG. 8 is a non-limiting example of a bar graph showing the effect ofthe addition of collagen gel (Col), AM extract AME, or collagen gelmixed with AM extract (Col+AME) on the suppression of TGF-β promoteractivity. BSA was used as a control.

FIG. 9 is a non-limiting example of a bar graph comparing the effect oftreatment with AME, HA, or HA+AME, compared to a control assay with BSAalone, on the suppression of TGFβ1 activity. The promoter activity isdisplayed as relative luciferase units (RLU).

FIG. 10 is a non-limiting example of an analysis of hyaluronan MW Rangesin AM Extracts of various AM extracts, separated by agarose gelelectrophoresis. Amniotic membrane extracted by buffer A, B, C weretreated with or without hyaluronidase and electrophoretically separatedby a 0.5% agarose gel.

FIG. 11 is a non-limiting example of an analysis of hyaluronan MW Rangesin AM Extracts of various AM extracts, separated by agarose gelelectrophoresis. Amniotic membrane extracted by buffer PBS were treatedwith or without hyaluronidase (10 units/ml in Tris-HCl, pH 7.5, 150 mMNaCl) for 2 hr at 37° C. and run through 0.5 agarose gels. HA: positivehyaluronic acid control; L: AM extract after low speed centrifugation;H: AM extract after high speed centrifugation.

FIG. 12 is a non-limiting example of a photograph of a western blotdemonstrating that the inter-α-trypsin inhibitor (IαI) is present in AMExtracts. IαI was present in AM extract A and C although the signal ofbikunin was very weak (˜39 kDa). Prior to transfer to the western blot,the extract was separated on a 4-15% denatured acrylamide gel.

FIG. 13 is a non-limiting example of an immunoblot demonstrating thatthe inter-α- trypsin inhibitor (IαI) is present in the AM extracts evenafter low (LS) or high speed (HS) centrifugation.

FIG. 14 is a non-limiting example of an immunoblot of TSG-6 (TumorNecrosis Factor-Stimulated Gene 6), either with (+) or without (−)hyaluronidase treatment. The samples included total AM extract withoutcentrifugation (T), AM Extract after extraction in isotonic low saltbuffer (buffer A); high salt buffer (B); or 4 M guanidine HCl (C); asdetailed in Example 2. TSG-6 was present in the total extract, buffer Aextract, and buffer C extract. The addition of hyaluronidase did notappear to alter the TSG-6 level in the extracts.

FIG. 15 is a non-limiting example of an immunoblot analysis of thedeglycosylation of TSG-6 in AM. AM extract A, B, and C were treated with(+) or without 20 units/ml PNGase F at 37° C. for 3 hours. Glycosylationof TSG-6 in AM was then analyzed by western blot. The cell lysate ofhuman corneal fibroblast (HCF) was used as a positive control.

FIG. 16 is a non-limiting example of an immunoblot analysis of potentialTSG-6 complexes in AM by digestion with Chondroitin Sulfate ABC lyase.AM extract A, B, and C were treated without (−) or with (+) 1 unit/mlABC lyase at 37° C. for 2 hours. The possible disruption of TSG-6complexes was then analyzed by western blot using an anti-TSG-6 antibodyRAH-1: 1:1000.

FIG. 17 is a non-limiting example of an immunoblot of potential TSG-6complexes in AM by digestion with Chondroitin Sulfate ABC lyase. This isthe same experiment as shown in FIG. 16 except that a different TSG-6antibody was used. Here, the anti-TSG-6 antibody was obtained from R & DSystems (cat# MAB2104).

FIG. 18 is a non-limiting example of an immunoblot demonstrating thepresence of Pentraxin (PTX3) in AM, using a rat monoclonal anti-PTX3antibody obtained from Alexis Biochemicals. HCF: human cornealfibroblast, T, A, B, C: AM extract Total, A, B, C, respectively; HAse,Hyaluronidase.

FIG. 19 is a non-limiting example of an immunoblot demonstrating thepresence of TSP-1 in AM. The monomeric TSP-1 (180 kDa) and the putativetrimeric TSP-1 (540 kDa) are indicated. The positive control, TSP-1, waspurified from human platelets (Calbiochem, Cat#605225) and loaded as 100ng/lane.

FIG. 20 is a non-limiting example of an immunoblot demonstrating thepresence of Smad 7 in AM. AM was extracted with PBS or urea (2M urea in50 mM Tris-HCl, pH 7.5). 20 μg of total protein was loaded for eachextract. Smad 7 was detected with goat anti-human Smad 7 (AF2029,1:1000, R & D Systems). Smad 7 migrated as a band of 51 kDa.

DETAILED DESCRIPTION OF THE INVENTION Compositions

Described herein are purified compositions that exert a number ofphysiologically significant effects in mammalian cells and intactmammalian tissues. The purified compositions comprise at least fourcomponents:

-   -   Cross-linked high molecular weight hyaluronan (HA);    -   Tumor necrosis factor-stimulated gene 6 (TSG-6);    -   Pentraxin (PTX-3); and    -   Thrombospondin (TSP-1).

Additional components may also be included in purified compositions thathave these four components, including: Smad7.

Any or all of the components of the purified compositions describedherein can be prepared from a human amniotic material, including humanamniotic jelly preparations and extracts (as described herein), humanamniotic membrane preparations and extracts (as described herein), andhuman amniotic stroma preparations and extracts (as described herein).

Together, these four components (with or without Smad7) can suppress TGFβ promoter activity; increase apoptosis in macrophages; decreaseproliferation, decrease migration, and increase apoptosis of humanvascular endothelial cells; decrease viability of human fibroblasts;decrease inflammation; and prevent apoptosis of epithelial cells exposedto storage and injury.

Hyaluronic acid (HA) is a natural sugar found in the synovial jointfluid, the vitreous humor of the eye, the cartilage, blood vessels,extra-cellular matrix, skin, and umbilical cord. The cross-linking of HAcan be through a covalent bound to another molecule, such as a protein.For example, HA can be covalently bound to the heavy chain ofinter-α-trypsin inhibitor. The ratio of protein to HA in the AMpreparations and purified compositions described herein can be less thanabout 200:1, less than about 100:1, less than about 50:1, or less thanabout 10:1.

TSG-6 is a hyaluronan binding protein that plays a role in extracellularmatrix remodeling, cell proliferation, and leucocyte migration. TSG-6can form a complex with the serine protease inhibitor inter-α-inhibitor.PTX-3 (Pentraxins) are Ca²⁺ dependent ligand binding proteins that havea pentameric discoid structure and are present in plasma. TSP-1(Thrombospondin I) is a homotrimeric glycoprotein having a potentanti-angiogenic and other biological activities. TSP-1 is secreted intothe extracellular matrix by a variety of cell types.

These components can be obtained from any suitable source. For example,at least one of the components can be obtained from human tissues, suchas amniotic membrane, amniotic jelly, amniotic stroma, or a combinationthereof. At least one of the components can be obtained from commercialsources. At least one of the components can be isolated from atransgenic organism. The protein sequences can have a similarity of atleast 90%, 93%, 95%, 97%, 99% or 99.5% to the human protein sequence.The components can be purified, substantially purified, partiallypurified, or can also be present in crude extracts. The components canalso be prepared from mammalian amniotic membrane tissues, as each ofthe four components is present in amniotic membrane tissues.

In additional aspects, the protein Smad7 is also present in thecomposition. The Smad7 can be obtained from any suitable source, such asfrom amniotic membrane, from a commercial source, isolated from atransgenic organism. The Smad7 protein can be purified, substantiallypurified, partially purified, or can be present in a crude extract.

AM Preparations derived from Placental Material

In some aspects, at least one of the components HA, TSG-6, PTX-3, TSP-1,optionally Smad7 can be obtained from preparations of amniotic membrane.Alternatively, crude amniotic membrane preparations containing thecombination of HA, TSG-6, PTX-3, TSP-1 and optionally Smad7 can beprepared. Exemplary methods of preparing various AM preparations aredescribed herein.

Human placental material can be obtained, for example, from sources suchas Bio-Tissue, Inc. (Miami, Fla.) and Baptist Hospital (Miami, Fla.)(under IRB approval). The tissue is typically obtained in either a freshor frozen state. The tissue can be washed to remove excess storagebuffer, blood, or contaminants. The excess liquid can be removed, forexample, using a brief centrifugation step, or by other means. Thetissue can be frozen, using, for example, liquid nitrogen or othercooling means, to facilitate the subsequent homogenization. The sourceof the AM tissue can be a human. However, other sources of AM tissue,such as bovine or porcine AM tissue, can be used.

The AM can be used to prepare the composition. AM preparations caninclude components or portions purified from or extracted from intactAM, AM stromal matrix, HA, AM jelly, and inter-alpha trypsin inhibitor(HA-ITI)). If desired, certain components of the AM preparation can beisolated from the preparation at any time during the process. Forexample, an extract enriched for a specific protein or set of AMproteins can be isolated from the preparation. After homogenization ofthe tissue, the larger particles can be separated out, or they can beleft in the preparation. The preparation can be dried, if desired. Anexemplary preparation method is described in Example 1.

The compositions can also be obtained from AM jelly. AM jelly can beobtained from the fresh AM tissue, or can be obtained before or afterthe freezing process. The AM jelly can be frozen, and can also befreeze-ground following the procedure for AM preparations as describedherein. The jelly can be centrifuged, and can also be lyophilized.

In additional embodiments, a composition made substantially from thestromal layer is prepared. To prepare this composition, the stromallayer is separated from the layer of fresh, frozen, thawed, or otherwisetreated AM membrane. The stromal removal can occur, for example, byenzymatic methods, mechanical methods, or by other means. The stromallayer material can be fresh or frozen. The stromal material can beground or freeze-ground following the procedure for AM preparations asdescribed herein. If desired, the stromal matrix material can becentrifuged, and can also be lyophilized.

The tissue can be frozen prior to the grinding process. The freezingstep can occur by any suitable cooling process. For example, the tissuecan be flash-frozen using liquid nitrogen. Alternatively, the materialcan be placed in an isopropanol/dry ice bath or can be flash-frozen inother coolants. Commercially available quick freezing processes can beused. Additionally, the material can be placed in a freezer and allowedto equilibrate to the storage temperature more slowly, rather than beingflash-frozen. The tissue can be stored at any desired temperature. Forexample, −20° C. or −80° C. or other temperatures can be used forstorage.

Pulverizing the tissue while frozen, rather than grinding the tissueprior to freezing, is one optional method for preparing the tissue.Alternatively, fresh, partially thawed, or thawed tissue can be used inthe grinding step. The tissue (fresh, frozen, or thawed) can then besliced into pieces of a desired size with a suitable device, such as ascalpel, then ground to fine particles using a BioPulverizer (BiospecProducts, Inc., Bartlesville, Okla.) or other suitable devices, andhomogenized with a homogenization device such as a Tissue Tearor(Biospec Products, Inc., Dremel, Wis., in a suitable solution. Exemplarysolutions include but are not limited to phosphate buffered saline(PBS), DMEM, NaCl solution, and water. The pH of the solution can beadjusted as needed. In some embodiments, the pH range is from about 5.5or 6.0 to about 8.5. In some embodiments, the frozen tissue is ground ina solution having a pH of between about 6.3, about 6.6, or about 7.0 toabout 7.4, about 7.6, or about 7.8.

Any suitable buffer or liquid can be used to prepare the formulations.Example 2 examines the use of various extraction buffers (high salt, lowsalt, PBS, etc.) on total protein content and HA in the preparation(Table 1). Example 2 further examines the levels of the specificproteins TSG-6 (FIG. 14), PTX-3 (FIG. 18), TSP-1 (FIG. 19), and Smad7(FIG. 20) using several extraction methods.

The homogenate can then be mixed at any suitable speed, temperature, orother parameters. The mixing can occur, for example, at a temperaturerange of from about 1° C., or 3° C., to about 6° C., 10° C., 15° C., or20° C. In some embodiments, the mixing occurs at about 4° C. Thehomogenate can be mixed, for example, from less than about 1 minute, 10minutes, or 20 minutes to about 1, 2, 3 or more hours.

The homogenate can then be centrifuged to remove any remaining largeparticulates, if desired. The centrifugation can be performed using anysuitable range of time, temperature, protein concentration, buffers, andspeed as desired. The centrifugation can occur, for example, at a rangeof about 1,000, 5,000, or 10,000×g to about 20,000×g. In someembodiments, the centrifugation occurs at about 15,000×g. Thecentrifugation can occur for a duration of from less than 1 minute, 5minutes, 10 minutes, 20 minutes, to about 40 minutes, 60 minutes, 1.5hours, or more. The supernatant can then be collected and stored inaliquots at −80° C. The total protein can be quantitated, if desired,using any suitable commercial protein analysis kit, such as a BCA assay(Pierce, Rockford, Ill.). Example 2, Table 1, and FIG. 13 describe theanalysis of AM preparations after low speed or high speedcentrifugation.

For biochemical characterization and purification, the above solutionscan be supplemented with protease inhibitors. An exemplary mixture ofprotease inhibitors is the following: 1 μg/mlaprotinin, 1 μg/mlleupeptin, 1 μg/ml pepstatin A, and 1 mM PMSF. Typically, however, aprotease inhibitor is not added to the preparation if it is to be addedto live cells or tissues.

The formulation can be tested to confirm the presence of specificcomponents or proteins. For example, the formulation can be tested forthe presence of molecules including but not limited to HA, TSG-6, PTX-3,TSP-1, Smad7, and the like. The formulation can also be tested toconfirm the absence of pathogens at any point during the preparationprocess.

AM preparations can be in a liquid, suspension, or lyophilized powder(e.g., ground or pulverized), or other forms. Antimicrobial agents suchas antibiotics or anti-fungal agents may be added. Other substances canbe added to the compositions to stabilize and/or preserve thecompositions. The material can be packaged and stored, for example, atroom temperature, or for example, at −20° C. or −80° C. prior to use.

In some embodiments, the preparation is present as a dry powderformulation. A dry powder formulation can be stored in a smaller volume,and may not require the same low temperature storage requirements tokeep the formulation from degrading over time. A dry powder formulationcan be stored and reconstituted prior to use. The dry powder formulationcan be prepared, for example, by preparing the freeze-ground AM tissueas described herein, then removing at least a portion of the water inthe composition. The excess water can be removed from the preparation byany suitable means. An exemplary method of removing the water is by useof lyophilization using a commercially available lyophilizer orfreeze-dryer. Suitable equipment can be found, for example, throughVirtis, Gardiner, N.Y.; FTS Systems, Stone Ridge, N.Y.; and SpeedVac(Savant Instruments Inc., Farmingdale, N.Y.). The amount of water thatis removed can be from about 5%, 10%, 20%, 30% to about 60, 70, 80, 90,95 or 99% or more. In some embodiments, substantially all of the excesswater is removed. The lyophilized powder can then be stored. The storagetemperature can vary from less than about −196° C. −80° C., −50° C., or−20° C. to more than about 23° C. If desired, the powder can becharacterized (weight, protein content, etc) prior to storage.

The lyophilized powder can be reconstituted in a suitable solution orbuffer prior to use. Exemplary solutions include but are not limited toPBS, DMEM, and BSS. The pH of the solution can be adjusted as needed.The concentration of the AM can be varied as needed. In some proceduresa more concentrated preparation is useful, whereas in other procedures,a solution with a low concentration of AM is useful. Additionalcompounds can be added to the composition. Exemplary compounds that canbe added to the reconstituted formulation include but are not limited topH modifiers, buffers, collagen, HA, antibiotics, surfactants,stabilizers, proteins, and the like. The lyophilized AM powder can alsobe added to a prepared cream, ointment or lotion to result in thedesired concentration.

Additional components can be added to the composition as desired. Insome embodiments, water soluble or powdered AM preparations can be mixedwith an ECM component such as collagen, fibrin, or HA.

Collagen is a major structural protein found in the body. It providessupport for tissues, connects tissue to bone, and provides the structureof the body. When the body is in the healing process, collagen plays arole in helping to build a cellular structure. Hyaluronic acid is anatural sugar found in the synovial joint fluid, the vitreous humor ofthe eye, the cartilage, blood vessels, extra-cellular matrix, skin, andumbilical cord. Fibrin is a protein involved in the clotting of blood.

Water-soluble AM preparation can be mixed with collagen, fibrin or withHA. Similarly, lyophilized powder AM preparation can be mixed withcollagen, fibrin or HA. Collagen, fibrin and HA can be are suitabledelivery vehicles, as AM preparations mixed with collagen or HA wereshown to exert a suppressive effect upon TGF β promoter activity.Although AM preparations were mixed with collagen gel and HA gel in theexperiments described herein, any soluble forms (e.g., liquid) ofcollagen and HA or other ECM components (e.g., fibrin) can be used. Thecollagen, fibrin or HA can be derived from any suitable source organism.When collagen, fibrin or HA are added, the ratio of these compounds toAM can be varied as desired. For example, a ratio of AM to collagen (orfibrin or HA) of less than about 0.001:1, 0.01:1, 0.05:1, or 0.1:1, toabout 1:1, 1.5:1, 2:1, 5:1, 10:1, 100:1 or 1000:1 or more can be used.

Collagen gel can be prepared, for example, by diluting the stocksolution (4 mg/ml) with 0.1 N acetic acid and by mixing it withappropriate volume ratios of 20× DMEM or suitable buffer, and 1 N NaOH,as described in Example 1. The collagen in the preparation can bepresent, for example, at a range of from less than about 2 mg/ml to morethan about 4 mg/ml.

Various dilutions of high MW HA can be prepared, for example, bydiluting commercially prepared HA (Healon™ (10 mg HA/ml) (Pharmacia,LaJolla, Calif.) in DMEM or suitable buffer. Lyophilized powder andwater-soluble forms of AM preparations can be diluted in a solution suchas PBS, DMEM, or other solutions into the desired collagenconcentration. The HA in the preparation can be present, for example, ata range of from less than about 2 μg/ml to more than about 129 μg/ml.

The following procedures represent illustrative methods for preparingthe amniotic preparations and purified compositions described and usedherein.

Preparation of Preserved Human AM:

Human placenta was collected at elective cesarean delivery (Heiligenhauset al., Invest Ophthalmol Vis Sci. 42:1969-1974, 2001, Lee and Tseng, AmJ Ophthalmol. 123:303-312, 1997, Prabhasawat et al., Ophthalmology,104:974-985, 1997, Tseng et al., Arch Ophthalmol. 116:431-441, 1998).The AM was flattened onto nitrocellulose paper (Hybond N+, Amersham,England), with the epithelium surface up. The AM samples were stored at−80° C. in DMEM /glycerol 1:2 (v/v) until use.

Amniotic Membrane Extract Preparations

Fresh and frozen human placentas were obtained from Bio-tissue, Inc.(Miami, Fla.). The entire procedure for preparation of total human AMextracts (AME) was carried out aseptically so as to be used forsubsequent cell culture-based experiments. The AM was sliced into smallpieces to fit into the barrel of a BioPulverizer (Biospec Products,Inc., Bartlesville, Okla.), frozen in the liquid nitrogen, pulverizedinto a fine powder, and weighed. Cold 1× PBS buffer, pH 7.4, containingprotease inhibitors (protease inhibitor cocktail, P8340, Sigma, andsupplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM sodiumfluoride and 0.2 mM sodium vanadate) was added to the powder at 1:1(ml/g). The mixture was kept on ice and homogenized with a Tissue Tearor(Biospec Products, Inc., Dremel, Wis.) 5 times, 1 minute each, with a 2minute cooling interval. These water-soluble extracts were designated as“Total” AM extracts (AME).

Total AM extracts were divided into two 50 ml conical centrifuge tubes.One was centrifuged at high speed (HS, 48,000×g) and the other one wascentrifuged at a low speed (LS, 15,000×g) at 4° C. Aliquots of the HSand LS supernatant were transferred to sterile 1.5 ml Eppendorf tubesand were designated as AM/HS, AM/LS, respectively. Desired AM/HS sampleswere frozen at −20° C. for 1 h before lyophilization. The samples thenwere placed in the chamber of FreeZone (Labconco, Kansas City, Mo.) withholes on the cap. Samples were lyophilized at −50° C. at a vacuum of0.85 mBar for 5 hours. Before use, the samples were reconstituted withthe sterile distilled H₂O to the same volume. The same method was alsoused to prepare extracts from AM jelly, which was easily scraped fromthe adherent material on the AM stroma.

Total Soluble Human Amniotic Membrane and Amniotic Membrane JellyExtract Preparations

Frozen human placenta material was obtained from Bio-Tissue, Bio-tissue,Inc. (Miami, Fla.). The entire procedure for preparation of total humanAM extracts (AME) was carried out aseptically so as to be used forsubsequent cell culture-based experiments. The AM was sliced into smallpieces to fit into the barrel of a BioPulverizer (Biospec Products,Inc., Bartlesville, Okla.), frozen in the liquid nitrogen, pulverizedinto a fine powder, and weighed. Cold 1× PBS buffer, pH 7.4, containingprotease inhibitors (protease inhibitor cocktail, P8340, Sigma, andsupplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM sodiumfluoride and 0.2 mM sodium vanadate) was added to the powder at 1:1(ml/g). The mixture was kept on ice and homogenized with a Tissue Tearor(Biospec Products, Inc., Dremel, Wis.) 5 times, 1 minute each, with a 2minute cooling interval. These water-soluble extracts were designated as“Total” AM extracts (AME).

The total water-soluble extract was mixed for 1 hr at 4° C.,subsequently fractionated by two different speeds of centrifugation at4° C. for 30 min, i.e., 15000×g and 48000×g, and the resultantsupernatant was designated as L and H, respectively. Each supernatantwas divided into aliquots and stored at −80° C. This method was alsoused to prepare extracts from AM jelly, which was easily scraped fromthe adherent material on the AM stroma.

Total Soluble Human Amniotic Membrane and Amniotic Membrane JellyExtracts by Different Buffers and Fractionation methods

In examining preparations in different extraction buffers, the powder asprepared from above was weighed and mixed with Buffer A (Isotonic Lowsalt): 100 mM Tris-HCl, pH 7.6, 150 mM NaCl, 4 mM EDTA, 1% Triton X-100at the wet weight (g) of AM to the buffer (ml) at 1:1 ratio by stirringat 4° C. for 1 hr. After centrifugation at 48000×g, the resultant pelletwas subsequently extracted by Buffer B (high salt): 100 mM Tris-HCl, pH7.6, 1 M NaCl, 4 mM EDTA, 1% Triton X-100 by stirring at 4° C. for 1 hr.Again after centrifugation at 48000×g, the pellet was finally extractedby Buffer C (4 M guanidine hydrochloride): 100 mM sodium acetate, pH5.8, 4 M guanidine hydrochloride, 4 mM EDTA, 1%Triton X-100 by stirringat 4° C. for 24 hr. All the above three buffers were supplemented withthe following protease and phosphatase inhibitors: 1 μg/ml aprotinin, 1μg/ml leupeptins, 1 μg/ml pepstatin A, 0.5 mM PMSF, 50 μM sodiumfluoride and 0.2 μM sodium vanadate. The resultant supernatants,designated as Extract A, B, and C, respectively, were dialyzed againstthe dislysis buffer (50 mM Tris-HCl, pH7.5, 0.15 M NaCl) supplementedwith 0.5 mM PMSF at 4° C. for 6 hr and dialysis buffer was changedtwice, each with 500× (the volume ratio- dialysis buffer:samples). Afterdialysis, the volume of each sample was measured and adjusted to thesame volume with the dialysis buffer. The same method was also used toprepare extracts from AM jelly, which was the adherent material on theAM stroma that could be easily scraped off.

Preparation of Total Soluble Human Amniotic Membrane Extracts in PBS

The entire procedure for preparation of total soluble human AM extracts(T) was carried out aseptically so as to be used for subsequent cellculture-based experiments. Frozen human placenta was obtained fromBio-tissue, Inc. (Miami, Fla.), from which AM was retrieved. AM wassliced into small pieces to fit into the barrel of a BioPulverizer(Biospec Products, Inc., Bartlesville, Okla.), frozen in the liquidnitrogen, and then pulverized into a fine powder. The powder was weighedand mixed with cold PBS buffer (prepared by adding distilled H₂O to 1×PBS, pH7.4, from 10× PBS, cat#70011-044, Invitrogen, Carlsbad, Calif.)with protease inhibitors (protease inhibitor cocktail, P8340, Sigma, andsupplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM sodiumfluoride and 0.2 mM sodium vanadate) at 1:1 (ml/g). The mixture was keptin the ice and homogenized with a Tissue Tearor (Biospec Products, Inc.,Dremel, Wis.) for 5 times, 1 min each with a 2 min interval cooling.This water-soluble extract was designated as “Total” (T). The totalwater-soluble extract was mixed for 1 hr at 4° C., centrifuged at 4° C.for 30 min at 48000×g. The supernatant was divided into aliquots andstored at −80° C.

Preparation of Water-soluble AM Stromal Extracts

Using aseptic techniques, frozen human AM obtained from Bio-Tissue, Inc.(Miami, Fla.) was briefly washed 2-3 times with HBSS to remove theoriginal storage medium. The AM stroma was scraped by spatula, frozen inthe air phase of liquid nitrogen and grounded to fine particles byBioPulverizer (Biospec Products, Inc., Bartlesville, Okla.) followed byhomogenization on ice with Tissue Tearor (Biospec Products, Inc.,Dremel, Wis.) in PBS, pH 7.4, for 1 min. The homogenate was mixed byrotation for 1 h and centrifuged at 14,000×g for 30 min at 4° C. Thesupernatant in PBS was then collected, and stored in aliquots at −80° C.The protein concentration was determined by BCA assay. Thiswater-soluble protein extract, designated as amniotic stromal extract(ASE), was used for experiments described herein.

AM Stromal Extract Preparation

The complete procedure for preparation of protein extracts was carriedout aspectically. Frozen human AM obtained from Bio-Tissue (Miami, Fla.)was briefly washed 2-3 times with HBSS (Invitrogen, Carlsbad, Calif.) toremove the storage medium. AM stroma was scraped from the stromal sideof the AM by spatula for AM stroma extract preparation. Human placentaas well as chorion obtained from Baptist Hospital (Miami, Fla.) wasrinsed 3 times with HBSS to remove blood. To prepare the water-solubleprotein extract, total AM, scraped AM stroma, stroma-removed AM,placenta, and chorion were each frozen in the air phase of liquidnitrogen and each ground to fine particles using a BioPulverizer(Biospec Products, Inc., Bartlesville, Okla.) followed by homogenizationon ice with Tissue Tearor (Biospec Products, Inc., Dremel, Wis.) in PBS(pH 7.4) for 1 min. Each homogenate was mixed for 1 hour and centrifugedat 14,000 g for 30 min at 40° C. Each supernatant (in PBS) was thencollected and stored in aliquots (0.5 ml) at 80° C. A BCA assay (Pierce,Rockford, Ill.) was used to quantitate the total protein in differentextracts.

Preparing Water-Soluble and Lyophilized Powder Forms of Human AMExtracts

In a typical procedure for preparing human AM extracts, the entireprocedure is carried out aseptically. Unless otherwise noted, the AMextracts can be handled at room temperature during the steps of theprocedure. First, fresh or frozen human AM is obtained, preferably fromBio-Tissue, Inc. (Miami, Fla.). Frozen AM is briefly washed 2-3 timeswith HBSS (Invitrogen, Carlsbad, Calif.) to remove the storage medium.Fresh human placenta or chorion is rinsed 3 times with HBSS to removeblood.

To prepare the water-soluble form of AM extracts, the AM (e.g., AMstroma, stroma-removed AM, placenta, chorion) is transferred to asterile 50 ml centrifuge tube and centrifuged at 4° C. for 5 min at5000×g to remove the excess fluid. The AM is weighed, transferred to a100 mm or 150 mm sterile Petri dish, and frozen in the air phase of aliquid nitrogen container for 20 min to facilitate the subsequenthomogenization. The frozen AM is then sliced into small pieces with adisposable scalpel or ground to fine particles using a BioPulverizer(Biospec Products, Inc., Bartlesville, Okla.) or other suitable device,and homogenized with Tissue Tearor (Biospec Products, Inc., Dremel,Wis.), or other suitable device, in phosphate buffered saline (PBS) orDMEM without phenol red (Invitrogen, Carlsbad, Calif.) at neutral pH.For biochemical characterization and purification, the above solutionsare supplemented with the following proteinase inhibitors: 1 μg/mlaprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mM PMSF.However, if the extract is to be directly added to cell culture, noprotease inhibitor is added. The homogenate is mixed at 4° C. for 30 mMand centrifuged at 15,000×g for 30 mM. The supernatant (i.e., AMextract) is collected and stored in aliquots (0.5 ml) at −80° C. A BCAassay (Pierce, Rockford, Ill.) is used to quantitate the total proteinin each AM extract.

To prepare the lyophilized powder form of AM extracts, frozen AM isground to fine particles using a BioPulverizer (Biospec Products, Inc.,Bartlesville, Okla.), or other suitable device, and further homogenizedas described herein. Aliquots of approximately 0.5 ml are lyophilized bySpeedVac (Savant Instruments Inc., Farmingdale, N.Y.) at 4° C. for 4 hto decrease the weight from 280 mg to 32 mg (˜89% reduction). Thelyophilized powder is weighed and stored at −80° C. Before use, thelyophilized powder can be reconstituted in a suitable buffer.

To prepare AM stromal extracts, the AM stroma is scraped from thestromal surface of intact total AM leaving the basement membrane and theamniotic epithelium intact, and the frozen AM stroma is ground using aBioPulverizer as described herein. The stroma is extracted with PBS(e.g., 1.4 mg/ml) at a neutral pH at 4° C. for 30 min and centrifuged at15,000×g for 30 mM. The supernatant is collected and stored in aliquots(0.5 ml) at −80° C. A BCA assay (Pierce, Rockford, Ill.) is used toquantitate the total protein in the AM stromal extract.

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 3^(rd) ed., vol. 1-3, ed. Sambrook et al., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; andCurrent Protocols in Molecular Biology, ed. Ausubel et al., GreenePublishing and Wiley-Interscience, New York, 2003 (with periodicupdates). Various techniques for culturing animal cells are known in theart and are described in Culture of Animal Cells: A Manual of BasicTechnique, 4^(th) ed., R. Ian Freshney, Wiley-Liss, Hoboken, N.J., 2000,and Animal Cell Culture Techniques (Springer Lab Manual), M. Clynos,Springer—Verlag, New York, N.Y., 1998. Methods involving proteinanalysis and purification are also known in the art and are described inProtein Analysis and Purification: Benchtop Techniques, 2^(nd) ed., IanM. Rosenberg, Birkhauser, New York, N.Y., 2004.

Pharmaceutical Compositions

AM preparations can be formulated for administration purposes as anon-solid dosage form, for example, by combining with a delivery vehicleto create compositions such as solutions, drops, suspensions, pastes,sprays, ointments, oils, emulsions, aerosols, a coated bandage, a patch,creams, lotions, gels, and the like. The formulation used will dependupon the particular application. Gels are useful for administering thecompositions because they allow better retention of the activeingredient at the site of introduction, allowing the active ingredientto exert its effect for a longer period of time before clearance of theactive ingredient. Alternatively, AM preparations can be formulated asextended-release solid dosage forms (including oral dosage forms). Adescription of exemplary pharmaceutically acceptable carriers orvehicles and diluents, as well as pharmaceutical formulations, isprovided herein and can also be found in Remington's PharmaceuticalSciences, a standard text in this field, and in USP/NF.

Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art. A summary of pharmaceuticalcompositions described herein may be found, for example, in Remington:The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins; 1999), herein incorporatedby reference in their entirety.

In certain embodiments, the compositions include a pharmaceuticallyacceptable diluent(s), excipient(s), or carrier(s). In addition, the AMpreparations and purified compositions described herein can beadministered as pharmaceutical compositions in which AM preparations andpurified compositions described herein are mixed with other activeingredients, as in combination therapy. In some embodiments, thepharmaceutical compositions may include other medicinal orpharmaceutical agents, carriers, adjuvants, such as preserving,stabilizing, wetting or emulsifying agents, solution promoters, saltsfor regulating the osmotic pressure, and/or buffers. In addition, thepharmaceutical compositions can also contain other therapeuticallyvaluable substances.

A pharmaceutical composition, as used herein, refers to a mixture of aAM preparations and purified compositions described herein with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. In practicing the methods of treatment or use providedherein, therapeutically effective amounts of AM preparations andpurified compositions described herein are administered in apharmaceutical composition to a mammal having a disease, disorder, orcondition to be treated. In some embodiments, the mammal is a human. Atherapeutically effective amount can vary widely depending on theseverity of the disease, the age and relative health of the subject, thepotency of the compound used and other factors. The compounds can beused singly or in combination with one or more therapeutic agents ascomponents of mixtures.

Topical Formulations

Formulations of the AM preparations and purified compositions describedherein include those suitable for topical administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredients which can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated, the particular mode of administration.

Suspensions may contain suspending agents as, for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, and mixtures thereof.

Typical compositions described herein include a wide variety of physicalforms. These include, but are not limited to, solutions, lotions,creams, oils, gels, sticks, sprays, ointments, balms, shampoo, andpastes. Generally, such carrier systems can be described as beingsolutions, emulsions, gels, solids, and aerosols. The compositions maybe applied topically to the skin, or may be applied in the form of atransdermal delivery device, such as a microneedle, a patch, bandage, orgauze pad known in the art.

The ointments, pastes, creams and gels may contain, in addition to theAM preparations and purified compositions described herein, excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the AM preparations andpurified compositions described herein, excipients such as lactose,talc, silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatileunsubstituted hydrocarbons, such as butane and propane.

Solvents are generally employed in the preparation of suitable topicalcompositions. Such solvents can either be aqueous or organic based. Thesolvent must be capable of having dispersed or dissolved therein theactive ingredients while not being irritating to the animal beingtreated. Water forms the basis for all aqueous solvents, while suitableorganic solvents include propylene glycol, butylene glycol, polyethyleneglycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitolesters, 1,2,6-hexanetriol, ethanol, isopropanol, butanediol, andmixtures thereof. Solvents can be included in the overall composition inamounts ranging from 0.1% to 99% and preferably from 2.0% to 75%. Insome embodiments, the compositions are produced in the form of anemollient-containing composition. A wide variety of suitable emollientsare known and may be used herein.

In some embodiments, the compositions are formulated as lotionscontaining from about 0.01% to 10% of the AM preparations and purifiedcompositions described herein. In other embodiments, the compositionsare formulated in a solution carrier system as a cream. A creamcomposition would preferably comprise from about 0.1% to 15% andpreferably from 1% to 5% of the the AM preparations and purifiedcompositions described herein. Lotions and creams can be formulated asemulsions as well as solutions. The compositions may also beadministered in liquid form, including in the form of liposomessuspended in liquid, as in the different type of sprays available inthis industry.

In other embodiments, the active ingredients are formulated asointments. Suitable ointments may comprise simple bases of animal orvegetable oils, or semi-solid hydrocarbons (oleaginous). Suitableointments may also comprise absorption ointment bases which absorb waterto form emulsions. Ointment carriers may also be water soluble. Anointment may comprise from 1% to 99% of an emollient plus to about 0.1%to 99% of a thickening agent.

The proportion of the AM preparations and purified compositionsdescribed herein in the compositions can vary from between about 0.01wt. % to about 100 wt. %, more preferably from about 0.1 wt. % to about99.9 wt. %, and especially from about 1.0 wt. % to about 99.0 wt. %.

“Carriers” or “vehicles” preferably refer to carrier materials suitablefor topical administration and include any such materials known in theart, such as any liquid, gel solvent, liquid diluent, solubilizer, orthe like, which is non-toxic, and which does not interact with othercomponents of the composition in a deleterious manner. Examples ofsuitable carriers for use herein include water, silicone, liquid sugars,waxes, oils, petroleum jelly, and a variety of other materials.

In some embodiments, the carrier or vehicle includes one or moresolvents, oils, surfactants, humectants, thickening agents,antioxidants, chelating agents, buffers, and preservatives.

Examples of solvents include C2-C10 alcohols, such as hexanol,cyclohexanol, benzyl alcohol, 1,2-butanediol, glycerol, and amylalcohol; C5-C 10 hydrocarbons such as n-hexane, cyclohexane, andethylbenzene; C4-C10 aldehydes and ketones, such as heptylaldehyde,cyclohexanone, and benzylaldehyde; C4-C10 esters, such as amyl acetateand benzyl propionate; ethereal oils, such as oil of eucalyptus, oil ofrue, cumin oil, limonene, thymol, and 1-pinene; halogenated hydrocarbonshaving 2-8 carbon atoms, such as 1-chlorohexane, 1-bromohexane, andchlorocyclohexane.

Examples of oils comprise fats and oils such as olive oil andhydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons suchas liquid paraffin, ceresin, and squalane; fatty acids such as stearicacid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol,lanolin alcohol, and hexadecanol; and esters such as isopropylmyristate, isopropyl palmitate and butyl stearate.

Examples of surfactants include anionic surfactants such as sodiumstearate, sodium cetyl sulfate, polyoxyethylene laurylether phosphate,sodium N-acyl glutamate; cationic surfactants such asstearyldimethylbenzylammonium chloride and stearyltrimethylammoniumchloride; ampholytic surfactants such as alkylaminoethylglycinehydrochloride solutions and lecithin; and nonionic surfactants such asglycerin monostearate, sorbitan monostearate, sucrose fatty acid esters,propylene glycol monostearate, polyoxyethylene oleylether, polyethyleneglycol monostearate, polyoxyethylene sorbitan monopalmitate,polyoxyethylene coconut fatty acid monoethanolamide, polyoxypropyleneglycol (e.g., the materials sold under the trademark “Pluronic”),polyoxyethylene castor oil, and polyoxyethylene lanolin.

Examples of humectants include glycerin, 1,3-butylene glycol, andpropylene glycol; examples of thickening agents include xanthan gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyethyleneglycol and sodium carboxymethyl cellulose; examples of antioxidantscomprise butylated hydroxytoluene, butylated hydroxyanisole, propylgallate, citric acid and ethoxyquin; examples of chelating agentsinclude disodium edetate and ethanehydroxy diphosphate; examples ofbuffers comprise citric acid, sodium citrate, boric acid, borax, anddisodium hydrogen phosphate; and examples of preservatives are methylparahydroxybenzoate, ethyl parahydroxybenzoate, dehydroacetic acid,salicylic acid and benzoic acid.

In certain embodiments, the carrier/vehicle is composed of the foregoingmaterials to achieve a controlled occlusion of the skin, therebyresulting in optimal enhancement of biologically active moietypenetration across the skin with minimal skin irritation. In certainembodiments, the carrier/vehicle may include a dispersing agent thataids in maintaining a particulate phase of the active ingredientsdispersed in the continuous phase. In other embodiments, non-ionicexcipients, such as lauric alcohol, propylene glycol monolaurate,myristyl lactate, lauryl lactate, or the like, facilitate dispersion.

The rate of biologically active moiety delivery across a dermal surfacecan be increased by transdermal delivery enhancers. Suitable transdermaldelivery enhancers include proton-accepting solvents such asdimethylsulfoxide and dimethylacetamide. Other suitable transdermaldelivery enhancers include 2-pyrrolidine, N,N-diethyl-m-toluamide, 1-dodecylazacycloheptan-2-one, N,N-dimethylformamide,N-methyl-2-pyrrolidine, terpenes, surfactants, and calciumthioglycolate.

Suitable dermal penetration enhancers include 1-5 carbon fatty acidesters of para-aminobenzoic acid, isopropyl palmitate, isopropylmyristate, ethanol, isobutyl alcohol, isobutyl alcohol, stearyl alcohol,glycerol, 2-pyrrolidone, urea, propylene glycol, oleic acid, palmiticacid, dimethyl sulfoxide, N,N-dimethyl acetamide, N,N-dimethylformamide, 2-pyrrolidone, 1-methyl-2-pyrrolidone,5-methyl-2-pyrrolidone, 1,5-dimethyl-2-pyrrolidone,1-ethyl-2-pyrrolidone, 2-pyrrolidone-5-carboxylic acid,N,N-dimethyl-m-toluamide, urea, ethyl acetate,1-dodecylazacycloheptan-2-one, oleic acid, imidazoline, butylurea, andpyrrolidone carboxylic acid esters.

Wetting agents, emulsifiers, surfactants, and lubricants, such as sodiumlauryl sulfate and magnesium stearate, as well as coloring agents,release agents, coating agents, sweetening, flavoring, and perfumingagents, preservatives and antioxidants can also be present in thecompositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite,and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

As appropriate compositions for topical application there may be citedall compositions usually employed for topically administeringtherapeutics, e.g., creams, jellies, dressings, shampoos, tinctures,pastes, ointments, salves, powders, liquid or semiliquid formulations,and the like. Application of said compositions may be by aerosol, e.g.,with a propellant such as nitrogen carbon dioxide, a freon, or without apropellant such as a pump spray, drops, lotions, or a semisolid such asa thickened composition which can be applied by a swab. In particularcompositions, semisolid compositions such as salves, creams, pastes,jellies, ointments, and the like will conveniently be used.

Ophthalmic Formulations

The AM preparations and purified compositions described herein can beadministered in a variety of ways, including all forms of local deliveryto the eye. Additionally, the AM preparations and purified compositionsdescribed herein can be administered systemically, such as orally orintravenously. The AM preparations and purified compositions describedherein can be administered topically to the eye and can be formulatedinto a variety of topically administrable ophthalmic compositions, suchas solutions, suspensions, gels or ointments. Thus, “ophthalmicadministration” encompasses, but is not limited to, intraocularinjection, subretinal injection, intravitreal injection, periocularadministration, subconjuctival injections, retrobulbar injections,intracameral injections (including into the anterior or vitreouschamber), sub-Tenon's injections or implants, ophthalmic solutions,ophthalmic suspensions, ophthalmic ointments, ocular implants and ocularinserts, intraocular solutions, use of iontophoresis, incorporation insurgical irrigating solutions, and packs (by way of example only, asaturated cotton pledget inserted in the fornix).

A composition comprising the AM preparations and purified compositionsdescribed herein can illustratively take the form of a liquid where theagents are present in solution, in suspension or both. Typically whenthe composition is administered as a solution or suspension a firstportion of the agent is present in solution and a second portion of theagent is present in particulate form, in suspension in a liquid matrix.In some embodiments, a liquid composition may include a gel formulation.In other embodiments, the liquid composition is aqueous. Alternatively,the composition can take the form of an ointment.

Useful compositions can be an aqueous solution, suspension orsolution/suspension, which can be presented in the form of eye drops. Adesired dosage can be administered via a known number of drops into theeye. For example, for a drop volume of 25 administration of 1-6 dropswill deliver 25-150 pl of the composition. Aqueous compositionstypically contain from about 0.01% to about 50%, more typically about0.1% to about 20%, still more typically about 0.2% to about 10%, andmost typically about 0.5% to about 5%, weight/volume of the AMpreparations and purified compositions described herein.

Typically, aqueous compositions have ophthalmically acceptable pH andosmolality. “Ophthalmically acceptable” with respect to a formulation,composition or ingredient typically means having no persistentdetrimental effect on the treated eye or the functioning thereof, or onthe general health of the subject being treated. Transient effects suchas minor irritation or a “stinging” sensation are common with topicalophthalmic administration of agents and consistent with the formulation,composition or ingredient in question being “ophthalmically acceptable.”

Useful aqueous suspension can also contain one or more polymers assuspending agents. Useful polymers include water-soluble polymers suchas cellulosic polymers, e.g., hydroxypropyl methylcellulose, andwater-insoluble polymers such as cross-linked carboxyl-containingpolymers. Useful compositions can also comprise an ophthalmicallyacceptable mucoadhesive polymer, selected for example fromcarboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

Useful compositions may also include ophthalmically acceptablesolubilizing agents to aid in the solubility of components of the AMpreparations and purified compositions described herein. The term“solubilizing agent” generally includes agents that result in formationof a micellar solution or a true solution of the agent. Certainophthalmically acceptable nonionic surfactants, for example polysorbate80, can be useful as solubilizing agents, as can ophthalmicallyacceptable glycols, polyglycols, e.g., polyethylene glycol 400, andglycol ethers.

Useful compositions may also include one or more ophthalmicallyacceptable pH adjusting agents or buffering agents, including acids suchas acetic, boric, citric, lactic, phosphoric and hydrochloric acids;bases such as sodium hydroxide, sodium phosphate, sodium borate, sodiumcitrate, sodium acetate, sodium lactate andtris-hydroxymethylaminomethane; and buffers such as citrate/dextrose,sodium bicarbonate and ammonium chloride. Such acids, bases and buffersare included in an amount required to maintain pH of the composition inan ophthalmically acceptable range.

Useful compositions may also include one or more ophthalmicallyacceptable salts in an amount required to bring osmolality of thecomposition into an ophthalmically acceptable range. Such salts includethose having sodium, potassium or ammonium cations and chloride,citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfateor bisulfite anions; suitable salts include sodium chloride, potassiumchloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

Other useful compositions may also include one or more ophthalmicallyacceptable preservatives to inhibit microbial activity. Suitablepreservatives include mercury-containing substances such as merfen andthiomersal; stabilized chlorine dioxide; and quaternary ammoniumcompounds such as benzalkoniuin chloride, cetyltrimethylammonium bromideand cetylpyridinium chloride.

Still other useful compositions may include one or more ophthalmicallyacceptable surfactants to enhance physical stability or for otherpurposes. Suitable nonionic surfactants include polyoxyethylene fattyacid glycerides and vegetable oils, e.g., polyoxyethylene (60)hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenylethers, e.g., octoxynol 10, octoxynol 40.

Still other useful compositions may include one or more antioxidants toenhance chemical stability where required. Suitable antioxidantsinclude, by way of example only, ascorbic acid and sodium metabisulfite.

Aqueous suspension compositions can be packaged in single-dosenon-reclosable containers. Alternatively, multiple-dose reclosablecontainers can be used, in which case it is typical to include apreservative in the composition.

The ophthalmic composition may also take the form of a solid articlethat can be inserted between the eye and eyelid or in the conjunctivalsac, where it releases the AM preparations and purified compositionsdescribed herein. Release is to the lacrimal fluid that bathes thesurface of the cornea, or directly to the cornea itself, with which thesolid article is generally in intimate contact. Solid articles suitablefor implantation in the eye in such fashion are generally composedprimarily of polymers and can be biodegradable or non-biodegradable.

Injectable Formulations

Formulations suitable for intramuscular, subcutaneous, or intravenousinjection may include physiologically acceptable sterile aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection may also contain additives such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the growth ofmicroorganisms can be ensured by various antibacterial and antifungalagents, such as parabens, chlorobutanol, phenol, sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections, AM preparations and purified compositionsdescribed herein may be formulated in aqueous solutions, inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, physiological saline buffer, or other suitable solutions. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art. For other parenteral injections, appropriateformulations may include aqueous or nonaqueous solutions, preferablywith physiologically compatible buffers or excipients. Such excipientsare generally known in the art.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Transdermal Formulations

Transdermal formulations described herein may be administered using avariety of devices which have been described in the art. For example,such devices include, but are not limited to, U.S. Pat. Nos. 3,598,122,3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636,3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084,4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303,5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and6,946,144, each of which is specifically incorporated by reference inits entirety.

The transdermal dosage forms described herein may incorporate certainpharmaceutically acceptable excipients which are conventional in theart. In one embodiments, the transdermal formulations described hereininclude at least three components: (1) a formulation as describedherein; (2) a penetration enhancer; and (3) an aqueous adjuvant. Inaddition, transdermal formulations can include additional componentssuch as, but not limited to, gelling agents, creams and ointment bases,and the like. In some embodiments, the transdermal formulation canfurther include a woven or non-woven backing material to enhanceabsorption and prevent the removal of the transdermal formulation fromthe skin. In other embodiments, the transdermal formulations describedherein can maintain a saturated or supersaturated state to promotediffusion into the skin.

Formulations suitable for transdermal administration of AM preparationsand purified compositions described herein may employ transdermaldelivery devices and transdermal delivery patches and can be lipophilicemulsions or buffered, aqueous solutions, dissolved and/or dispersed ina polymer or an adhesive. Such patches may be constructed forcontinuous, pulsatile, or on demand delivery of pharmaceutical agents.Still further, transdermal delivery of the AM preparations and purifiedcompositions described herein can be accomplished by means ofiontophoretic patches and the like. Additionally, transdermal patchescan provide controlled delivery of the composition. The rate ofabsorption can be slowed by using rate-controlling membranes or bytrapping the compound within a polymer matrix or gel. Conversely,absorption enhancers can be used to increase absorption. An absorptionenhancer or carrier can include absorbable pharmaceutically acceptablesolvents to assist passage through the skin. For example, transdermaldevices are in the form of a bandage comprising a backing member, areservoir containing the compound optionally with carriers, optionally arate controlling barrier to deliver the compound to the skin of the hostat a controlled and predetermined rate over a prolonged period of time,and means to secure the device to the skin.

Solid Oral Dosage Forms

The pharmaceutical solid dosage forms described herein can include oneor more pharmaceutically acceptable additives such as a compatiblecarrier, binder, filling agent, suspending agent, flavoring agent,sweetening agent, disintegrating agent, dispersing agent, surfactant,lubricant, colorant, diluent, solubilizer, moistening agent,plasticizer, stabilizer, penetration enhancer, wetting agent,anti-foaming agent, antioxidant, preservative, or one or morecombination thereof. In still other aspects, using standard coatingprocedures, such as those described in Remington's PharmaceuticalSciences, 20th Edition (2000).

Compressed tablets are solid dosage forms prepared by compacting thebulk blend of the formulations described herein. In various embodiments,compressed tablets which are designed to dissolve in the mouth willinclude one or more flavoring agents. In other embodiments, thecompressed tablets will include a film surrounding the final compressedtablet. In some embodiments, the film coating can provide a delayedrelease of the composition from the formulation. In other embodiments,the film coating aids in patient compliance (e.g., Opadry® coatings orsugar coating). Film coatings including Opadry® typically range fromabout 1% to about 3% of the tablet weight. In other embodiments, thecompressed tablets include one or more excipients.

A capsule may be prepared, for example, by placing the bulk blend of theformulation of the composition described herein, inside of a capsule. Insome embodiments, the formulations (non-aqueous suspensions andsolutions) are placed in a soft gelatin capsule. In other embodiments,the formulations are placed in standard gelatin capsules or non-gelatincapsules such as capsules comprising HPMC. In other embodiments, theformulation is placed in a sprinkle capsule, wherein the capsule may beswallowed whole or the capsule may be opened and the contents sprinkledon food prior to eating. In some embodiments, the therapeutic dose issplit into multiple (e.g., two, three, or four) capsules. In someembodiments, the entire dose of the formulation is delivered in acapsule form.

In various embodiments, the particles of the composition and one or moreexcipients are dry blended and compressed into a mass, such as a tablet,having a hardness sufficient to provide a pharmaceutical compositionthat substantially disintegrates within less than about 30 minutes, lessthan about 35 minutes, less than about 40 minutes, less than about 45minutes, less than about 50 minutes, less than about 55 minutes, or lessthan about 60 minutes, after oral administration, thereby releasing theformulation into the gastrointestinal fluid.

In another aspect, dosage forms may include microencapsulatedformulations. In some embodiments, one or more other compatiblematerials are present in the microencapsulation material. Exemplarymaterials include, but are not limited to, pH modifiers, erosionfacilitators, anti-foaming agents, antioxidants, flavoring agents, andcarrier materials such as binders, suspending agents, disintegrationagents, filling agents, surfactants, solubilizers, stabilizers,lubricants, wetting agents, and diluents.

The pharmaceutical solid dosage forms including AM preparations andpurified compositions described herein can be further formulated toprovide a controlled release of the composition. Controlled releaserefers to the release of the composition from a dosage form in which itis incorporated according to a desired profile over an extended periodof time. Controlled release profiles include, for example, sustainedrelease, prolonged release, pulsatile release, and delayed releaseprofiles. In contrast to immediate release compositions, controlledrelease compositions allow delivery of an agent to a subject over anextended period of time according to a predetermined profile. Suchrelease rates can provide therapeutically effective levels of agent foran extended period of time and thereby provide a longer period ofpharmacologic response while minimizing side effects as compared toconventional rapid release dosage forms. Such longer periods of responseprovide for many inherent benefits that are not achieved with thecorresponding short acting, immediate release preparations.

In some embodiments, the solid dosage forms described herein can beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the smallintestine of the gastrointestinal tract. The enteric coated dosage formmay be a compressed or molded or extruded tablet/mold (coated oruncoated) containing granules, powder, pellets, beads or particles ofthe active ingredient and/or other composition components, which arethemselves coated or uncoated. The enteric coated oral dosage form mayalso be a capsule (coated or uncoated) containing pellets, beads orgranules of the solid carrier or the composition, which are themselvescoated or uncoated.

In other embodiments, the formulations described herein are deliveredusing a pulsatile dosage form. A pulsatile dosage form is capable ofproviding one or more immediate release pulses at predetermined timepoints after a controlled lag time or at specific sites. Pulsatiledosage forms may be administered using a variety of pulsatileformulations known in the art. For example, such formulations include,but are not limited to, those described in U.S. Pat. Nos. 5,011,692,5,017,381, 5,229,135, and 5,840,329, each of which is specificallyincorporated by reference. Other pulsatile release dosage forms suitablefor use with the present formulations include, but are not limited to,for example, U.S. Pat. Nos. 4,871,549, 5,260,068, 5,260,069, 5,508,040,5,567,441 and 5,837,284, all of which are specifically incorporated byreference.

Many other types of controlled release systems known to those ofordinary skill in the art and are suitable for use with the formulationsdescribed herein. Examples of such delivery systems include, e.g.,polymer-based systems, such as polylactic and polyglycolic acid,plyanhydrides and polycaprolactone; porous matrices, nonpolymer-basedsystems that are lipids, including sterols, such as cholesterol,cholesterol esters and fatty acids, or neutral fats, such as mono-, di-and triglycerides; hydrogel release systems; silastic systems;peptide-based systems; wax coatings, bioerodible dosage forms,compressed tablets using conventional binders and the like. See, e.g.,Liberman et al., Pharmaceutical Dosage Forms, 2 Ed., Vol. 1, pp. 209-214(1990); Singh et al., Encyclopedia of Pharmaceutical Technology, 2^(nd)Ed., pp. 751-753 (2002); U.S. Pat. Nos. 4,327,725, 4,624,848, 4,968,509,5,461,140, 5,456,923, 5,516,527, 5,622,721, 5,686,105, 5,700,410,5,977,175, 6,465,014 and 6,932,983, each of which is specificallyincorporated by reference.

In some embodiments, pharmaceutical formulations are provided thatinclude particles of the compositions described herein and at least onedispersing agent or suspending agent for administration to a subject.The formulations may be a powder and/or granules for suspension, andupon admixture with water, a substantially uniform suspension isobtained.

The aqueous suspensions and dispersions described herein can remain in ahomogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005edition, chapter 905), for at least 4 hours. The homogeneity should bedetermined by a sampling method consistent with regard to determininghomogeneity of the entire composition. In one embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 1 minute. In another embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 45 seconds. In yet another embodiment, anaqueous suspension can be re-suspended into a homogenous suspension byphysical agitation lasting less than 30 seconds. In still anotherembodiment, no agitation is necessary to maintain a homogeneous aqueousdispersion.

Suitable viscosity enhancing agents for the aqueous suspensions ordispersions described herein include, but are not limited to, methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, Plasdon® S-630, carbomer,polyvinyl alcohol, alginates, acacia, chitosans and combinations thereofThe concentration of the viscosity enhancing agent will depend upon theagent selected and the viscosity desired.

In addition to the additives listed above, the liquid formulations canalso include inert diluents commonly used in the art, such as water orother solvents, solubilizing agents, and emulsifiers. Exemplaryemulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodiumdoccusate, cholesterol, cholesterol esters, taurocholic acid,phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

It is to be appreciated that there is overlap between the above-listedadditives used in the aqueous dispersions or suspensions describedherein, since a given additive is often classified differently bydifferent practitioners in the field, or is commonly used for any ofseveral different functions. Thus, the above-listed additives should betaken as merely exemplary, and not limiting, of the types of additivesthat can be included in formulations described herein. The amounts ofsuch additives can be readily determined by one skilled in the art,according to the particular properties desired.

Intranasal Formulations

Intranasal formulations are known in the art and are described in, forexample, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452, each ofwhich is specifically incorporated by reference. Formulations can beprepared according to these and other techniques well-known in the artare prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, fluorocarbons, and/or other solubilizing ordispersing agents known in the art. See, for example, Ansel, H. C. etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed.(1995). These compositions and formulations can be prepared withsuitable nontoxic pharmaceutically acceptable ingredients. Theseingredients are known to those skilled in the preparation of nasaldosage forms and some of these can be found in REMINGTON: THE SCIENCEAND PRACTICE OF PHARMACY, 21st edition, 2005, a standard reference inthe field. The choice of suitable carriers is highly dependent upon theexact nature of the nasal dosage form desired, e.g., solutions,suspensions, ointments, or gels. Nasal dosage forms generally containlarge amounts of water in addition to the active ingredient. Minoramounts of other ingredients such as pH adjusters, emulsifiers ordispersing agents, preservatives, surfactants, gelling agents, orbuffering and other stabilizing and solubilizing agents may also bepresent.

For administration by inhalation, the compositions described herein maybe in a form as an aerosol, a mist or a powder. Pharmaceuticalcompositions described herein are conveniently delivered in the form ofan aerosol spray presentation from pressurized packs or a nebuliser,with the use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the AM preparations and purified compositions described hereinand a suitable powder base such as lactose or starch.

Other Formulations

The AM preparations and purified compositions described herein may alsobe formulated in rectal compositions such as enemas, rectal gels, rectalfoams, rectal aerosols, suppositories, jelly suppositories, or retentionenemas, containing conventional suppository bases such as cocoa butteror other glycerides, as well as synthetic polymers such aspolyvinylpyrrolidone, PEG, and the like. In suppository forms of thecompositions, a low-melting wax such as, but not limited to, a mixtureof fatty acid glycerides, optionally in combination with cocoa butter isfirst melted.

Methods of Dosing and Treatment REgimens

The compositions can be administered by any suitable technique.Typically, the compositions will be administered directly to a targetsite (e.g., ocular surface, skin). The administration of formulations tothe ocular surface is well known in the art. If delivery of AMpreparations to the skin is desired, topical administration can be used.An injectable composition is also envisioned. Administration can also beparenteral (e.g., subcutaneous). Other methods of delivery, e.g.,liposomal delivery, diffusion from a device impregnated with thecomposition, and microemulsion-based transdermal delivery in bothcosmetic and pharmaceutical applications, are known in the art.

The compositions containing the AM preparations and purifiedcompositions described herein can be administered for prophylacticand/or therapeutic treatments. In therapeutic applications, thecompositions are administered to a patient already suffering from adisease or condition, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease or condition. Amountseffective for this use will depend on the severity and course of thedisease or condition, previous therapy, the patient's health status,weight, and response to the drugs, and the judgment of the treatingphysician. It is considered well within the skill of the art for one todetermine such therapeutically effective amounts by routineexperimentation (including, but not limited to, a dose escalationclinical trial).

In prophylactic applications, compositions containing the AMpreparations and purified compositions described herein are administeredto a patient susceptible to or otherwise at risk of a particulardisease, disorder or condition. Such an amount is defined to be a“prophylactically effective amount or dose.” In this use, the preciseamounts also depend on the patient's state of health, weight, and thelike. It is considered well within the skill of the art for one todetermine such prophylactically effective amounts by routineexperimentation (e.g., a dose escalation clinical trial). When used in apatient, effective amounts for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the compounds may beadministered chronically, that is, for an extended period of time,including throughout the duration of the patient's life in order toameliorate or otherwise control or limit the symptoms of the patient'sdisease or condition.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds may be givencontinuously; alternatively, the dose of drug being administered may betemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). The length of the drug holiday can varybetween 2 days and 1 year, including by way of example only, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days,180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or365 days. The dose reduction during a drug holiday may be from 10%-100%,including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained. Patients can, however, require intermittenttreatment on a long-term basis upon any recurrence of symptoms.

The amount of a given agent that will correspond to such an amount willvary depending upon factors such as the particular compound, disease orcondition and its severity, the identity (e.g., weight) of the subjector host in need of treatment, but can nevertheless be routinelydetermined in a manner known in the art according to the particularcircumstances surrounding the case, including, e.g., the specific agentbeing administered, the route of administration, the condition beingtreated, and the subject or host being treated. In general, however,doses employed for adult human treatment will typically be in the rangeof 0.02-5000 mg per day, preferably 1-1500 mg per day. The desired dosemay conveniently be presented in a single dose or as divided dosesadministered simultaneously (or over a short period of time) or atappropriate intervals, for example as two, three, four or more sub-dosesper day.

The pharmaceutical composition described herein may be in unit dosageforms suitable for single administration of precise dosages. In unitdosage form, the formulation is divided into unit doses containingappropriate quantities of one or more compound. The unit dosage may bein the form of a package containing discrete quantities of theformulation. Non-limiting examples are packaged tablets or capsules, andpowders in vials or ampoules. Aqueous suspension compositions can bepackaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers can be used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection may be presented in unitdosage form, which include, but are not limited to ampoules, or in multidose containers, with an added preservative.

The daily dosages appropriate for the AM preparations and purifiedcompositions described herein are from about 0.01 to 2.5 mg/kg per bodyweight. An indicated daily dosage in the larger mammal, including, butnot limited to, humans, is in the range from about 0.5 mg to about 100mg, conveniently administered in divided doses, including, but notlimited to, up to four times a day or in extended release form. Suitableunit dosage forms for oral administration include from about 1 to 50 mgactive ingredient. The foregoing ranges are merely suggestive, as thenumber of variables in regard to an individual treatment regime islarge, and considerable excursions from these recommended values are notuncommon. Such dosages may be altered depending on a number ofvariables, not limited to the activity of the compound used, the diseaseor condition to be treated, the mode of administration, the requirementsof the individual subject, the severity of the disease or conditionbeing treated, and the judgment of the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD50 (the dose lethal to 50% of the population) and the ED50 (thedose therapeutically effective in 50% of the population). The dose ratiobetween the toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio between LD50 and ED50. Compoundsexhibiting high therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED50 with minimal toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

Combination Treatments

The compositions and methods described herein may also be used inconjunction with other well known therapeutic reagents that are selectedfor their particular usefulness against the condition that is beingtreated. In general, the compositions described herein and, inembodiments where combinational therapy is employed, other agents do nothave to be administered in the same pharmaceutical composition, and may,because of different physical and chemical characteristics, have to beadministered by different routes. The determination of the mode ofadministration and the advisability of administration, where possible,in the same pharmaceutical composition, is well within the knowledge ofthe skilled clinician. The initial administration can be made accordingto established protocols known in the art, and then, based upon theobserved effects, the dosage, modes of administration and times ofadministration can be modified by the skilled clinician.

The particular choice of compounds used will depend upon the diagnosisof the attending physicians and their judgment of the condition of thepatient and the appropriate treatment protocol. The compounds may beadministered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,depending upon the nature of the disease, disorder, or condition, thecondition of the patient, and the actual choice of compounds used. Thedetermination of the order of administration, and the number ofrepetitions of administration of each therapeutic agent during atreatment protocol, is well within the knowledge of the skilledphysician after evaluation of the disease being treated and thecondition of the patient.

It is known to those of skill in the art that therapeutically-effectivedosages can vary when the drugs are used in treatment combinations.Methods for experimentally determining therapeutically-effective dosagesof drugs and other agents for use in combination treatment regimens aredescribed in the literature. For example, the use of metronomic dosing,i.e., providing more frequent, lower doses in order to minimize toxicside effects, has been described extensively in the literature.Combination treatment further includes periodic treatments that startand stop at various times to assist with the clinical management of thepatient.

For combination therapies described herein, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the disease orcondition being treated and so forth. In addition, when co-administeredwith one or more biologically active agents, the compound providedherein may be administered either simultaneously with the biologicallyactive agent(s), or sequentially. If administered sequentially, theattending physician will decide on the appropriate sequence ofadministering protein in combination with the biologically activeagent(s).

In any case, the multiple therapeutic agents may be administered in anyorder or even simultaneously. If simultaneously, the multipletherapeutic agents may be provided in a single, unified form, or inmultiple forms (by way of example only, either as a single pill or astwo separate pills). One of the therapeutic agents may be given inmultiple doses, or both may be given as multiple doses. If notsimultaneous, the timing between the multiple doses may vary from morethan zero weeks to less than four weeks. In addition, the combinationmethods, compositions and formulations are not to be limited to the useof only two agents; the use of multiple therapeutic combinations arealso envisioned.

It is understood that the dosage regimen to treat, prevent, orameliorate the condition(s) for which relief is sought, can be modifiedin accordance with a variety of factors. These factors include thedisorder from which the subject suffers, as well as the age, weight,sex, diet, and medical condition of the subject. Thus, the dosageregimen actually employed can vary widely and therefore can deviate fromthe dosage regimens set forth herein.

The pharmaceutical agents which make up the combination therapydisclosed herein may be a combined dosage form or in separate dosageforms intended for substantially simultaneous administration. Thepharmaceutical agents that make up the combination therapy may also beadministered sequentially, with either therapeutic compound beingadministered by a regimen calling for two-step administration. Thetwo-step administration regimen may call for sequential administrationof the active agents or spaced-apart administration of the separateactive agents. The time period between the multiple administration stepsmay range from, a few minutes to several hours, depending upon theproperties of each pharmaceutical agent, such as potency, solubility,bioavailability, plasma half-life and kinetic profile of thepharmaceutical agent. Circadian variation of the target moleculeconcentration may also determine the optimal dose interval.

In addition, the AM preparations and purified compositions describedherein also may be used in combination with procedures that may provideadditional or synergistic benefit to the patient. By way of exampleonly, patients are expected to find therapeutic and/or prophylacticbenefit in the methods described herein, wherein pharmaceuticalcomposition of a compound disclosed herein and /or combinations withother therapeutics are combined with genetic testing to determinewhether that individual is a carrier of a mutant gene that is known tobe correlated with certain diseases or conditions.

The AM preparations and purified compositions described herein andcombination therapies can be administered before, during or after theoccurrence of a disease or condition, and the timing of administeringthe composition containing a compound can vary. Thus, for example, thecompounds can be used as a prophylactic and can be administeredcontinuously to subjects with a propensity to develop conditions ordiseases in order to prevent the occurrence of the disease or condition.The compounds and compositions can be administered to a subject duringor as soon as possible after the onset of the symptoms. Theadministration of the compounds can be initiated within the first 48hours of the onset of the symptoms, preferably within the first 48 hoursof the onset of the symptoms, more preferably within the first 6 hoursof the onset of the symptoms, and most preferably within 3 hours of theonset of the symptoms. The initial administration can be via any routepractical, such as, for example, an intravenous injection, a bolusinjection, infusion over 5 minutes to about 5 hours, a pill, a capsule,transdermal patch, buccal delivery, and the like, or combinationthereof. A compound is preferably administered as soon as is practicableafter the onset of a disease or condition is detected or suspected, andfor a length of time necessary for the treatment of the disease, suchas, for example, from about 1 month to about 3 months. The length oftreatment can vary for each subject, and the length can be determinedusing the known criteria. For example, the compound or a formulationcontaining the compound can be administered for at least 2 weeks,preferably about 1 month to about 5 years, and more preferably fromabout 1 month to about 3 years.

Kits/Articles of Manufacture

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. Such kits can includea carrier, package, or container that is compartmentalized to receiveone or more containers such as vials, tubes, and the like, each of thecontainer(s) including one of the separate elements to be used in amethod described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers can be formedfrom a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, andany packaging material suitable for a selected formulation and intendedmode of administration and treatment. A wide array of formulations ofthe compounds and compositions provided herein are contemplated as are avariety of treatments for any disease, disorder, or condition.

For example, the container(s) can include one or more AM preparationsand purified compositions described herein, optionally in a compositionor in combination with another agent as disclosed herein. Thecontainer(s) optionally have a sterile access port (for example thecontainer can be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). Such kits optionallycomprising a compound with an identifying description or label orinstructions relating to its use in the methods described herein.

A kit will typically may include one or more additional containers, eachwith one or more of various materials (such as reagents, optionally inconcentrated form, and/or devices) desirable from a commercial and userstandpoint for use of the AM preparations and purified compositionsdescribed herein. Non-limiting examples of such materials include, butnot limited to, buffers, diluents, filters, needles, syringes; carrier,package, container, vial and/or tube labels listing contents and/orinstructions for use, and package inserts with instructions for use. Aset of instructions will also typically be included.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Alabel can be used to indicate that the contents are to be used for aspecific therapeutic application. The label can also indicate directionsfor use of the contents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions can be presentedin a pack or dispenser device which can contain one or more unit dosageforms containing a compound provided herein. The pack can for examplecontain metal or plastic foil, such as a blister pack. The pack ordispenser device can be accompanied by instructions for administration.The pack or dispenser can also be accompanied with a notice associatedwith the container in form prescribed by a governmental agencyregulating the manufacture, use, or sale of pharmaceuticals, whichnotice is reflective of approval by the agency of the form of the drugfor human or veterinary administration. Such notice, for example, can bethe labeling approved by the U.S. Food and Drug Administration forprescription drugs, or the approved product insert. Compositionscontaining a compound provided herein formulated in a compatiblepharmaceutical carrier can also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition.

Methods of Treatment

The AM preparations and purified compositions described herein have manyuses including research and clinical applications. Based on the resultsdescribed herein, the AM preparations and purified compositionsdescribed herein can be applied to tissues or cells to achieve a desiredmodulation of physiology. AM preparations and purified compositionsdescribed herein can further be added to cell cultures or tissuecultures to achieve a desired effect (as described herein).

Suppression of TGF Promoter Activity

The AM preparations and purified compositions described herein suppressof TGF-β1 promoter activity as shown herein; thus the AM preparationsand purified compositions described herein can be used foranti-scarring, anti-inflammatory, and anti-angiogenic therapies. Thefetal portion of the frozen amniotic membrane has a significantly higheranti-scarring effect than that of fresh amniotic membrane; the placentalportion of the frozen amniotic membrane also has a significantly higheranti-scarring effect than the fresh amniotic membrane (Example 1).Therefore, the frozen AM, either the placental or fetal portion, showedmore potent suppressive effects in TGF-β than the fresh AM. Thissuppressive effect mediated by total AM extract obtained from frozen AMwas dose-dependent over a range of 0.4 to 125 μg/ml. Furthermore, such asuppressive effect could not be substituted by high MW HA alone(exceeding 100× of equivalent AM extract), and was lost after digestionwith hyaluronidase, suggesting that it was mediated by a complex betweenHA-IαI. Centrifugation at low or high speed did not affect thesuppressive effect significantly. However, subsequent lyophilization andreconstitution produced a more potent suppressive effect. Additionally,the overall suppressive effect of AM was more potent than that of AMjelly.

TGF-β is the prototypic cytokine that is involved in tissueinflammation, in addition to wound healing and scar formation. SeeBorder, et al., J. Clin. Invest., 90:1-7 (1992); Grande, Proc. Soc. Exp.Biol. Med., 214:27-40 (1997); Jester, et al., Prog. Retin. Eye Res.,18(3):311-356 (1999); and Marek, et al., Med. Sci. Monit.,8(7):RA145-151 (2002). Mammalian cells express three different TGF-βs:TGF-β1, TGF-β2, and TGF-β3. TGF-β is the most potent cytokine promotingmyofibroblast differentiation by up-regulating expression of α-SMA,integrin α5β1, and EDA domain-containing fibronectin (Fn) in a number ofcell types, including fibroblasts. See Tseng, et al., Ocular Surface J.,2(3):177-187 (2004); Ronnov-Jessen, et al., Lab. Invest., 68:696-707(1993); Verbeek, et al., Am. J. Pathol., 144:372-82 (1994); Hales, etal., Curr. Eye Res., 13:885-90(1994); Jester, et al., Cornea, 15:505-16(1996); Serini, et al., J. Cell. Biol., 142:873-81 (1998); Grande, Proc.Soc. Exp. Biol. Med., 214(1):27-40 (1997); and Jester, et al., Prog.Retin. Eye. Res., 18:311-56 (1999). TGF-β also up-regulates theexpression of such matrix components as collagens and proteoglycans,down-regulates proteinase and matrix metalloproteinases, andup-regulates their inhibitors. Collectively, these actions result inincreased cell-matrix interactions and adhesiveness, as well asdeposition and formation of scar tissue. See Tseng, et al., OcularSurface J., 2(3):177-187 (2004); Grande, Proc. Soc. Exp. Biol. Med.,214(l):27-40 (1997); Jester, et al., Prog. Retin. Eye. Res., 18:311-56(1999); and Lawrence, Eur. Cytokine Netw., 7:363-74 (1996).

TGF-βs exert their actions via binding with TGF-beta receptors (TGF-βRs)on the cell membrane. In human cells, there are three TGF-βRs, namelyTGF-βR type I (TGF-βRI), type II (TGF-βRII), and type III (TGF-βRIII).TGF-βs, serving as ligands, bind with a serine, threonine kinasereceptor complex made of TGF-βRI and TGF-βRII; such a binding isfacilitated by TGF-βRIII, which is not a serine, threonine kinasereceptor. See Tseng, et al., Ocular Surface J., 2(3):177-187 (2004); andMassague, et al., Genes and Development., 14:627-44 (2000). Binding withTGF-βRII activates TGF-βRI, which is responsible for directphosphorylation of a family of effector proteins known as Smads, whichmodulate transcription of a number of target genes, including thosedescribed herein, participating in scar formation. See Tseng, et al.,Ocular Surface J., 2(3):177-187 (2004); Massague, et al., Genes andDevelopment., 14:627-44 (2000); and Derynck, et al., Biochem. Biophys.Acta., 1333:F105-F150 (1997).

Suppression of TGF-β can be achieved by neutralizing antibodies to TGF-βand agents that intercede the signaling mediated by TGF-β such asdecorin. See Shahi, et al., Lancet, 339:213-214 (1992); Petroll, et al.,Curr. Eye Res., 1739:736-747 (1998); Yamaguchi, et al., Nature,346(6281):281-284 (1990); and Logan, et al., Exp. Neurol., 159:504-510(1999). Most of the literature has shown suppression of TGF-β beingachieved at the level of modulating the TGF-β activation, binding withits receptor, or its signal transduction. It has been shown thatamniotic membrane can achieve such an inhibition at the level oftranscription, i.e., to turn off transcription of TGF-β genes. Inparticular, amniotic membrane has been shown to suppress TGF-β signalingin human corneal and limbal fibroblasts, and human conjunctival andpterygium body fibroblasts. See Tseng, et al., J Cell Physiol.,179:325-335 (1999); and Lee, et al., Curr. Eye Res., 20(4):325-334(2000).

Application of the AM preparations and purified compositions describedherein can be used to lower the production or activity of TGF-β. Severaltypes of AM compositions, such as AME (total human AM extract), the AMEsupernatant after centrifugation, AM jelly, and AM stroma were preparedas detailed in Example 1. The effect of various buffers, such as PBS,low salt buffer, high salt buffer, and guanidine HCl on TGF-β activitywas examined. Additionally, the effect of various freeze-grindingprocedures on TGF-β activity was examined.

The suppression of TGF-β activity was lost after hyaluronidasedigestion, demonstrating that the suppressive effect may be mediated bya HA-related complex (FIG. 3). The suppressive effect was not recoveredby addition of HA. The centrifugation step did not alter the suppressionof TGF-β activity, in either AME or AM jelly extracts (FIG. 5).Lyophilization enhanced the suppression of TGF-β activity of both AMEand jelly extract (FIG. 6). FIG. 8 and FIG. 9 demonstrate that collagenand HA, when added to AME, can enhance the suppression of TGF-βactivity. Accordingly, addition of collagen and HA to the AM-basedcompositions may be useful to treat various diseases involving TGF-β.

As shown herein, TGF-β is downregulated by the disclosed compositions.Accordingly, the compositions described herein can be used to treatdiseases related to TGF-β is downregulation, such as angiogenesis, woundhealing, and tissue inflammation.

AM Preparations and Purified Compositions Described herein can PreventApoptosis

The AM preparations and purified compositions described herein can beused to prevent, lessen, or treat apoptosis in tissues. In someembodiments, the AM preparations and purified compositions describedherein can decrease or prevent apoptosis in tissues that have beeninjured. This anti-apoptotic effect demonstrates that the compositionscan be used to prolong the life of organs being stored prior totransplant. The compositions can also be used to treat or prevent damageduring and after surgical procedures. Example 3 demonstrates theanti-apoptotic effect of AM extract using a murine model of eye damage.Mouse eyes were collected and damaged either by enzymatic treatment orby mechanical injury, AM extract was administered, and the effect oncellular damage was determined, using an assay that measures apoptoticdamage to the nucleus. Incubation with AM extract was found to decreasethe levels of apoptosis.

Because of the anti-apoptotic effects exerted by AM preparation, AMpreparations and compositions are expected to be useful for preservingtissues (e.g., cornea) before transplantation. The addition of AMpreparations to tissues that are being stored can be helpful inlessening cellular damage due to the storage process. For example, theAM preparations and purified compositions described herein can be usedto decrease the amount of degradation that occurs in a tissue that isbeing stored prior to transplantation or surgical procedures. The AMpreparations and purified compositions described herein can be added tothe storage medium, with or without collagen and/or HA. Stored tissuessuch as eyes, organs, skin, and the like can benefit from the decreasedcellular apoptosis that occurs when an AM composition is added.

Once a donor tissue is harvested, it is typically stored in a storagemedium until transplantation. The compositions can be added to thestorage medium to prevent cellular apoptosis. For example, thecompositions can be added to storage media for preserving limbalepithelial stem cells. Similarly, AM preparation-containing compositionscan be added to cell culture medium or digestion medium to preventcellular (e.g., keratocyte) apoptosis. Because studies described hereinshow that incubation of AM preparation during dispase digestion (atreatment which mimics surgical and pathological insults such as excimerablation in PRK and recurrent corneal erosion, respectively)significantly reduced apoptosis of both epithelial cells andkeratocytes, the compositions can also be administered to an eyereceiving mechanical scraping or excimer laser photoablation to attemptto reduce keratocyte apoptosis, and hence reduce corneal haze. Asanother example, AM preparation-containing formulations can also be usedin surgical conditions or diseases such as recurrent corneal erosion orkeratoconus where the basement membrane is dissolved to reduce thekeratocyte apoptosis.

The compositions and methods described herein are provided furtherdetail in the following examples. These examples are provided by way ofillustration and are not intended to limit the invention in any way.

AM Preparations and Purified Compositions Described herein can be usedto Culture Cells

The AM preparations can be used to produce a phenotypic reversal ofAMSCs from myofibroblasts to fibroblasts. The AM preparations andpurified compositions described herein can also be used to prevent orslow differentiation of various cell types. Many types of cells can betreated with the AM preparations. This method is particularly useful forexpanding cell cultures without causing differentiation of the cultureto unwanted cell types.

To examine the effect of AM on the cell differentiation process,myofibroblasts differentiated from AMSCs at passage 2 were subculturedonto the stromal matrix of AM, and compared to those subcultured oncollagen I-coated dish as a control. After 7 days of cultivation in DMEMwith 10% FBS, AMSCs on collagen I still maintained a myofibroblasticshape. In contrast, cells seeded on AM stromal matrix exhibited amixture of round, spindle, elongated, and dendritic shapes. Thus, AMpreparations have dedifferentiation abilities, and can be used to slowcell differentiation.

AM Preparations and Purified Compositions Described herein can be usedto Culture Tissue

The AM preparations and purified compositions described herein canadditionally be helpful in culturing biological tissues. For example, AMpowdered extract at 1 mg/ml can be added to cultured skin tissues orcorneal tissues.

AM Preparations and Purified Compositions Described herein can be usedto Prevent Organ Damage Arising from Organ Transplants

AM preparations and compositions can be added to organ storage media tolessen or prevent damage arising from organ transplants. For example,the AM preparations and purified compositions described herein can beadded to a storage medium to decrease the amount of damage that occursin a tissue that is being stored prior to transplantation. Storedtissues such as eyes, organs, skin, and the like can benefit from thedecreased cellular apoptosis that occurs when an AM composition isadded.

EXAMPLES Example 1 Suppressive Activities of Various Amniotic MembranePreparations Hyaluronidase Digestion

AM total water-soluble extracts (AME) prepared from frozen AM were mixedwith or without 10 units/ml hyaluronidase (Sigma #H1136) in the reactionbuffer (50 mM HEPES, pH7.5, 0.1 M NaCl, 1% Triton X-100, 0.1% BSAsupplemented with the above protease and phosphatase inhibitors for 2hours at 37° C. using a positive control of high MW HA (cat# H1876,Sigma) purified from human umbilical cords.

Cell Culture and TGF-β1 Promoter Suppression

When human corneal fibroblasts cultured on 100 mm plastic dish inDMEM/10% FBS reached 80% confluency (˜1.0×10⁶ cells), cells were washedtwice with DMEM/10% FBS. Adenoviruses-TGF-β1 promoter-luciferase(MOI=37.5) and Adeno-CMV-beta-gal (MOI=30) were added to the cultureplates with 10 ml of the fresh DMEM/10% FBS and cells were incubated at37° C. for 4 hours, and trypsinized for 5 minutes using 4 ml prewarmedtrypsin/EDTA. After trypsin/EDTA activity was neutralized with 8 ml ofDMEM/10% FBS, cells were collected into a 15 ml tube and centrifuged at1,500 rpm (˜600×g) for 5 min. After decanting the medium, cells wereresuspended in 15 ml DMEM/10% FBS, and cell viability was measured bytrypan blue stain. Viable 3×10⁴ cells were seeded on a plastic 24 wellor on the stromal surface of AM inserts. A total of 4 wells or insertswere prepared. Cells were then incubated at 37° C. in a CO2 incubatorfor 48 hours.

After carefully removing the growth medium from each well, cells wererinsed with 0.5 ml PBS at lease twice, taking care not to dislodgeattached cells. After removing as much as PBS in the well, 100 μl 1×lysis buffer was added to cover the cells, and cells were mechanicallyscraped and transferred to a microcentrifuge tube placed on ice. Celllysates were collected by vortexing for 10-15 sec and centrifuging at12,000×g for 15 sec at room temperature. The supernatant designated ascell lysate was stored at −80° C. prior to assaying for luciferaseactivities.

Suppression of TGF-β1 Promoter Activity by Different AM Extracts

In FIG. 1, compared to the plastic control (PL), both the placentalportion and the fetal portion of frozen amniotic membrane (FRO/P andFRO/F, respectively) showed significant suppression of TGF-β1 promoteractivity (each P<0.01). For the fresh placenta, the placental portion ofamniotic membrane (FRE/P) also exhibited a significant suppression ofTGF-β1 promoter activity (P<0.05). Nevertheless, the fetal portion ofthe fresh amniotic membrane (FRE/F) did not show any suppressive effect(P=0.5). These results indicated that the fetal portion of the freshamniotic membrane does not have the same anti-scarring effect as thefrozen counterpart. For the frozen amniotic membrane, the suppressiveeffect by the placental portion (FRO/P) was not significantly differentfrom that by the fetal portion (P=0.3). For the fresh amniotic membrane,the suppressive effect by the fetal portion (FRE/F) was notsignificantly from the placental portion (FRE/P) (P=0.1). For theplacental portion, the suppressive effect by the frozen amnioticmembrane (FRO/P) was significantly better than the fresh amnioticmembrane (FRE/P) (P<0.05). In the fetal portion, however, thesuppressive effect by the frozen amniotic membrane (FRO/F) was notsignificantly different than the suppressive effect of the freshamniotic membrane (FRE/F) (P=0.1).

Suppression of TGF-β1 Promoter Activity is Dose-Dependent and Lost afterDigestion with Hyaluronidase

The suppression of TGF-β1 promoter activity by total water-soluble AMextracts prepared from frozen AM obeyed a dose-responsive curve from0.04 to 125 μg/ml (FIG. 2). As shown by the promoter activity of TGF-β1and TGF-βRII, the suppressive effect of 25 μg/ml of total water-solubleAM extracts prepared from frozen AM was lost when pre-treated withhyaluronidase, indicating that such a suppressive effect was mediated byan HA-related complex (FIG. 3). It should be noted that 25 μg/ml AMextracts contained less than 0.78 μg/ml HA.

Lost Suppressive Effect from Hyaluronidase cannot be Recovered byAddition of HA

Although 100 μg/ml high MW HA alone showed a mild suppressive activity,its magnitude was still significantly less than 25 μg/ml AM extracts.Taken together, these data suggest that the suppressive effect of AMextracts was mediated by HA-linked complex, i.e., HA-IαI complex.

Soluble AME and Jelly Extracts Derived after Centrifugation Do NotChange the Suppressive effect on TGF-β1 Promoter Activities

Compared to the PBS control, HA, AM (Total, Low Speed, High speed) andJelly (Total, Low Speed, High Speed) showed suppression of TGF-β1promoter activation when normalized with beta-galatosidase activity. Pvalue indicated there was not statistically significant due to thevariation among the control group (data not shown). By comparing totalAME and two conditions of centrifuged soluble AME, results suggestedthat there was no significant difference. However, withoutcentrifugation of AME showed less suppression compare with low or highsoluble AME. Likewise, Jelly/T indicated less TGF-β suppressionactivities in comparison with Jelly/HS (FIG. 5).

Lyophilization Enhanced the Suppressive Effect of AME and Jelly Extract

Human corneal fibroblasts showed no change in cell morphology in thecontrol, HA alone, and low concentrations of AME or jelly extracts (Datanot shown). However, cells showed a marked change to slender and smallcells after the treatment with high concentration of AME and L/AME, asearly as 18 hrs after seeding (FIG. 6). Furthermore, the cell densityalso decreased. The above changes were even more dramatic in lyophilizedAME or L/AME than their non-lyophilized counterparts in AME or Jellyextracts, respectively (FIG. 6).

To examine whether the TGF-beta promoter was suppressed during AMEtreatment, luciferase assays were performed. Beta-galatosidase assay wasused as the transfection control. The result indicated that AME-125,L-AME-25, L-AME-125, L-Jelly-125 showed a significant differences ininhibiting TGF-beta 1 promoter activities, the percentage of inhibitionswere 86%(P<0.01), 55% (P<0.1), 95% (P<0.01), and 46% (P<0.1),respectively (FIG. 7). The data suggests that lyophilized form of AME orJelly at a high concentration of 125 μg/ml was more effective than thenon-lyophilized AME form. Although the lowest concentration ofcommercial HA (4 μg/ml) was close to the concentration HA (3.8 μ/gml) inAME/125, the effectiveness suppression in AME/125 is far more potentthan HA. (Data not shown) Furthermore, the AME form overall illustrateda better TGF-beta suppression than the Jelly form.

Suppression of TGF-β1 Promoter Activity by AM Extracts Mixed WithCollagen Gel or HA

A mixture of native type 1 collagen gel and water-soluble AM extract wasthen prepared. To prepare this mixture, collagen gel was first preparedby diluting a 4 mg/ml stock collagen solution prepared from rat tailtendon (BD Biosciences, San Jose, Calif.) with 0.1 N acetic acid andmixing it with a 1/20 volume ratio of 20× DMEM and 1 N NaOH. A collagengel formed after incubation at 37° C. Next, water-soluble AM extract(prepared as described herein) was diluted in DMEM to a concentration of25 μg/ml and then mixed with the collagen gel. The suppressive effect ofAM extract mixed in type 1 collagen gel was similar to that of AMextracts (AME) used alone, when compared to the control which was addedwith BSA alone (FIG. 8, p<0.01). Although collagen gel alone (Col) alsoshowed a similar suppressive activity when compared to the plasticcontrol (FIG. 8, p<0.01), addition of AME in collagen gel (Col+AME)resulted in further suppression (FIG. 8, p<0.01). When water-soluble AMextracts (AME) were mixed in HA gel, the suppressive effect on TGF β1promoter activity was better preserved as compared to HA alone (mixedwith BSA as a control) (FIG. 5, p<0.01) similar to that exerted by AMEalone (FIG. 9). Accordingly, an AM extract composition, or itscombination with collagen can be useful to suppress TGF β activity ineye tissue.

Example 2 Characterization of Amniotic Membrane Components Material andMethods

The concentration of proteins in each extract was quantitated by the BCAProtein Assay Kit (Pierce, Rockford, Ill.). The concentration ofhyaluronic acid (HA) in each extracts was assayed with Hyaluronic Acid(HA) Quantitative Test Kit (Corgenix, Westminster, Colo.) based on ELISAusing a standard curve provided by the manufacturer prepared by serialdilution of HA.

HA Molecular Weight Range Analysis by Hyaluronidase Digestion

The HA molecular weight ranges of the extracts were analysed by agarosegel electrophoresis according to the method described by Lee and Cowman(Lee H. G. and Cowman, M. K. An Agarose Gel Electrophoretic Method forAnalysis of Hyaluronan Molecular Weight Distribution. AnalyticalBiochemistry, 1994, 219, 278-287). The samples were subjected to 0.5%agarose gel electrophoresis followed by staining using 0.005% Stains-All(Sigma, cat#23096-0) in 50% ethanol. The gel was stained overnight undera light-protective cover at room temperature (Shorter staining periodsof 3-4 hr can also give acceptable results). HA was visualized as bluebands after destaining by transferring the gel to H₂O and exposed to theroom light for approximately 6 hr. The molecular weight standardsincluded lamda DNA-BstE II digested restriction fragments (cat# D9793,Sigma) ranging in MW from 0.9 to 5.7×10⁶. The authenticity of HA wasfurther verified by incubation of the extract with or without 10units/ml hyaluronidase (Sigma #H1136) in the reaction buffer (50 mMTris-HCl, pH7.5, 0.1 M NaCl, 1% Triton X-100, 0.1% BSA supplemented withthe above protease and phosphotase inhibitors) for 2 h at 37° C. using apositive control of high MW HA (cat# H1876, Sigma) purified from humanumbilical cords.

Western Blot Analyses

The above extracts were electrophoresized on 4-15% denatured acrylamidegels and transferred to the nitrocellulose membrane, and thenimmunoblotted with a rabbit anti-human inter-α-trypsin inhibitor (rabbitpolyclonal antibody (cat# A0301, DAKO at 1:1000), a rabbit anti-humanTSG-6 polyclonal antibody (provided by Dr. Tony Day at 1:1000 dilution),a rat monoclonal anti-PTX3 antibody (Alexis Biochemicals, ALX-804-464, 1μg/ml), an anti-thrombospondin-1 antibody obtained from Calbiochem (Cat#BA24), and a goat anti-human Smad 7 antibody (AF2029, 1:1000, R & DSystems). Imunoreactive protein bands were detected by Western Lighting™Chemiluminesence Reagent (PerkinElmer).

Results

Experiments showed that the observed suppressive effect on the TGF β1promoter activity was abolished when water-soluble AM extracts werepre-heated at 90° C. for 10 minutes, suggesting that the responsiblecomponent(s) most likely contained protein(s), of which the conformationis important.

Quantitation of HA and Proteins in AM Extracts

The results summarized in the Table below showed that all AM and jellyextracts contained both HA and proteins. In general, the weight ratiobetween proteins and HA was high in the Total Extract than thesupernatant (e.g., L and H for PBS, and A for Buffer A) aftercentrifugation for AM, suggesting that most protein-containing materialswere eliminated by centrifugation. However, this trend was not noted inAM Jelly, suggesting that AM extracts contained more proteins than Jelly(see T under PBS and T under A/B/C). The ratio between proteins and HAwas also increased from Extract A to Extracts B and C for both AM and AMjelly, further supporting that HA was mostly present in the solubleform, and vice versa proteins were found more in the water-insolublecomponents. Furthermore, HA was largely removed from AM Jelly aftercentrifugation in AB/C.

TABLE 1 Tissue AM Jelly Buffer PBS A/B/C PBS A/B/C Fraction T L H T A BC T L H T A B C Protein 8645 1370 1467 8645 2731 930 2698 3836 3645 35893836 3893 527 1364 (μg/ml) HA 75 62 44 60 74 7 35 80 90 96 129 94 2 7(μgml) Protein/ 115 22 33 144 37 133 77 48 41 37 30 41 264 195 HA[Note]: T: Total, L: the supernatant following the low speedcentrifugation of the total extract, H: the supernatant following thelow speed centrifugation of the total extract, A, B, C: Extracts, seetext.HA in Different AM Extracts Have Molecular Weights Greater than OneMillion Daltons

High molecular weight (>10⁶ daltons) of HA was present in the totalextracts and Extract A (FIG. 10). However, even higher MW of HA waspresent in Extract B, while HA was found in a narrow band with evenhigher MW in Extract C (FIG. 10). All of the HA-containing componentsdisappeared after hyaluoridase digestion, confirming that they indeedcontained HA. Compared to the positive control of HA obtained from Sigma(cat# H1136), a similar high molecular weight (>10⁶ daltons) of HA wasalso found in both supernatants obtained after low and high speeds ofcentrifugation (FIG. 11). Again these HA-containing bands disappearedafter hyaluonidase digestion. A similar result was obtained for AM jelly(not shown).

Inter-α-Trypsin Inhibitor (IαI) is Present in Different AM Extracts andIts Heavy Chains (HCs) Are Covalently Linked with HA

FIG. 12 showed that before digestion with hyaluronidase, free heavychains were present in different complexes, and a small amount of lightchain was also present (UTI or bikunin). However, in all extracts, i.e.,total and Extracts A, B, and C, there was also a covalently linkedcomplex between HA and heavy chains of IαI as the latter was releasedonly after hyaluronidase digestion. The same result was obtained inExtracts H and L obtained by two different speeds of centrifugation(FIG. 13).

Tumor Necrosis Factor-Stimulated Gene 6 (TSG-6) is also Present in AMExtracts

FIG. 14 showed that TSG-6 (˜38 kDa) was present in Total, Extract A andExtract C. In Extract A, there was a band of ˜38 kDa migrated close tothat of the purified TSG-6 (35 kD). The identity of other bands of ˜45and 55 kDa was unknown. Total AM extract (without centrifugation) “T”showed two bands (both above 35 kD), and the higher one (55 kD) thatwere found in Extract A (after centrifugation), while the lower one (45kD) was found in Extract C. All of these bands were not significantlyaltered when samples were treated with hyaluronidase (FIG. 14) or withF-glycosidase (FIG. 15). However, digestion with chondroitin sulfate ABClyase resulted in more noticeable 38 kD band using antibody RAH-1 (FIG.16) but not using antibody MAB2104 (FIG. 17).

Pentraxin (PTX-3) Is Exclusively Present in Water-soluble AM Extractsand Forms a Complex with HA

FIG. 18 showed that PTX3 could also be present in AM extracts and iscomplexed with HA in the water soluble extract A only.

Thrombospondin (TSP-1) Is Present in Different AM Extracts

FIG. 19 showed that all AM extracts had a high molecular weight band ofTSP-1 while the total extract (T) and Extract C also had some bandsbetween 35-120 kDa. Hyaluronidase digestion did not change the reactivepattern except some bands became a little stronger or weaker.

Smad7 Is Present in Mostly in Water-Insoluble AM Extracts

Smad 7 was found in both PBS extracts and urea extracts of AM (FIG. 20).

Example 3 Water-Soluble AM Extracts Prevent Cell Death of CornealEpithelial Cells (Basal Cells and Keratocytes) Induced by Storage and byInjuries Caused by Mechanical and Enzymatic Means Results

To demonstrate that AM extracts can prevent apoptosis in injuredtissues, the following experiment was performed using a murine model. Atotal of 22 mouse eye balls were enucleated, two of which wereimmediately embedded in OCT for frozen sections as a pretreatmentcontrol. The remaining of 20 eye balls were subdivided into threesubgroups, namely, 1) mechanical scraping (n=8), 2) dispose digestion(enzymatic) (n=6), and 3) without treatment control (n=6). For eachgroup, equal numbers of eye balls were preincubated at 4° C. for 24 h inthe presence (+) or absence (−) of 125 μg/ml AM extracts in keratinocyteserum-free medium (KSFM) with defined supplement (Gibco, Carlsbad,Calif.) prior to the treatments. At the end of the first 24 h ofincubation in KSFM +/- AM extract (prepared as described herein), 8eyeballs in Subgroup 1 were then subjected to mechanical scraping with asurgical blade, and were further divided into two groups (n=4 each) andincubated at 37° C. in KSFM +/− AM extract. Six eye balls in Subgroup 2were subjected to enzymatic digestion with 10 mg/ml Dispase II in KSFM+/− AM extract (n=3 each) for 18 h at 4° C. One eye ball from each groupwas embedded in OCT for frozen sections. The remaining two eye ballsfrom each group were incubated in KSFM +/−AM extract for another 24 hbefore analysis. For the non-treatment control (n=6), 3 eye balls eachwere incubated in KSFM +/− AM extract at 37° C. continuously for twodays; one eye ball was removed at the end of the first day while two eyeballs were removed at the end of 2 days.

The frozen sections from these eyeballs were subjected to TUNELstaining. In short, terminal deoxyribonucleotidyl transferase-mediatedFITC-linked dUTP nick-end DNA labeling (TUNEL) assay was performed usingDeadEnd™ fluorometric TUNEL system obtained from Promega (Madison, Wis.)according to the manufacturer's instructions. Sections were fixed in 4%formaldehyde for 20 min at room temperature and permeabilized with 1%Triton X-100. Samples were then incubated for 60 min at 37° C. withexogenous TdT and fluorescein-conjugated dUTP for repair of nicked3′-hydroxyl DNA ends. Cells were treated with DNase I as the positivecontrol, while the negative control was incubated with buffer lackingrTdT enzyme. The apoptotic nuclei were labeled with green fluorescence,and the nuclei were counterstained with DAPI as red fluorescence.

The water-soluble form of AM extract was prepared by the method forpreparing water-soluble AM extract. A BCA assay (Pierce, Rockford, Ill.)was used to quantitate the total protein in the AM extract.

The normal mouse eye ball showed a minimal amount of apoptosis only inthe superficial layer of the corneal epithelium of the uninjured controlbefore incubation in KSFM; no apoptosis was noted in the stromalkeratocytes. However, after 24 h incubation at 4° C. in KSFM, there wasa mild increase of apoptosis in keratocytes of the superficial stroma.Such an increase of keratocyte apoptosis was suppressed by AM extract.

The AM extract was also shown to reduce apoptosis after mechanicaldamage to the cells. After mechanical scraping, the mouse eye ballshowed a significant increase of keratocyte apoptosis. However,incubation with AM extract following mechanical scraping resulted in adecrease in keratocyte apoptosis.

The mouse eyes were also treated enzymatically to damage the cells.Dispase digestion at 4° C. for 18 h in KSFM resulted in a significantamount of apoptosis in not only keratocytes but also in epithelialcells; for the latter apoptosis was found to be present not only in thesuperficial epithelial cells, but also in the basal epithelial cells.The extent of epithelial and keratocyte apoptosis was far greater thanthat noted after mechanical scraping. Incubation of AM extract duringdispase digestion significantly reduced apoptosis of both epithelialcells and keratocytes. This is significant because dispase treatmentmimics the surgical (e.g., excimer ablation in PRK) and pathologicalinsults (e.g., recurrent corneal erosion) that can be directed to thebasement membrane. The results of this experiment demonstrate that theapplication of AM extract to tissues with damaged cells can be used toreduce or prevent cellular damage.

Example 4 Comparing the Relative Potency of Using Collagen or HA as aVehicle to Deliver Two Different AM Extracts

To determine the optimal concentration of the water-soluble andlyophilized forms of AM extracts (prepared by the methods describedherein for preparing water-soluble and lyophilized forms of AM extracts,respectively) and compare the relative potency between the two differentvehicles containing an appropriate concentration of each form of AMextracts in suppressing TGF β promoter activity and in promotingmacrophage apoptosis, respectively, these two forms of AM extracts arecompared by serial dilution in either type I collagen gel or HA andtheir protein concentrations are monitored accordingly. For type Icollagen gel, the protein concentration varies from 0.05 to 2 mg/ml; forHA gel, the concentration varies from 0.05 to 10 mg/ml. These seriallydiluted solutions or gels of these two forms of AM extracts arepre-coated on plastic dishes before human corneal fibroblasts are seededor added directly in DMEM with 10% FBS while cells are seeded on theplastic. The anti-scarring effect is measured by assaying the promoteractivity of TGF β1, β2, β3 and RII and comparing the promoter activityto the positive or negative controls where cells are seeded on plasticwith or without a given form of AM extracts (without the vehicle),respectively. The positive control, in which cells were seeded onplastic with DMEM plus 10% FBS, showed a high promoter activity. Incontrast, the negative control, in which cells were seeded on plasticwith DMEM plus 10% FBS but added with 25 μg/ml AM extracts, showed atleast 50% reduction of the promoter activity. Based on these controlvalues, the experimental groups using different concentrations of AMextracts mixed in either collagen gel or HA can be measured.

Once the most effective concentration of these two forms of AM extractsin either collagen or HA is determined, the results are verified byrepeating the experiment in serum-free DMEM with ITS added with 10 pg/mlto 5 ng/ml TGF β1. The anti-scarring effect is further correlated withsuppression of Smad-mediated signaling by immunocytolocalization ofSmads 2, 3 and 4 and a smooth muscle actin (α SMA), a marker formyofibroblasts (Gabbiani G., J Pathol. 200:500-503, 2003; Jester andPetroll, Prog Retin Eye Res. 18:311-356, 1999). Another positive controlis performed by adding 10 μg/ml neutralizing antibody to all threeisoforms of TGF β. The anti-inflammatory effect is similarly tested inmurine macrophages with or without activation by 200 U/ml IFN γ in DMEMwith ITS by measuring the extent of apoptosis using Cell Death DetectionELISAPLUS kit (Roche, Mannheim, Germany), and correlating the data withthose obtained by cell morphology, LIVE/DEAD assay (Molecular Probes,Carlsbad, Calif.), Hoechst-33342 nuclear staining, and TUNEL assay(Promega, Madison, Wis.) as recently reported (Li et al., Exp Eye Res.2005, In Press).

Example 5 Skin Lotion Composition Containing AM Extract

A skin lotion is prepared by the following method. 0.25 g methylhydroxybenzoate and 7.5 g gycerin are dissolved in 75 ml of water at150° F. 0.7 g sorbitan monolaurate, 0.7 g polysorbate 20, and 1.0 gcetostearyl alcohol are melted at 150° F. and are then compounded intothe solution. The mixture is allowed to cool while mixing. When themixture reaches a temperature below approximately 90° F., 4 ml of AMpreparations and purified compositions described herein is added whilemixing. A trace amount of fragrance is also added while mixing. Thelotion is packaged into 10 ml aliquots and stored at room temperature.

Example 6 Ophthalmic Solution Composition and Treatment of an EyeDisease

An opthalmic eye drop solution is prepared by mixing 100 mg of ground,lyophilized AM extract with 0.9 g of NaCl in 100 mL of purified waterand filtered using a 0.2 micron filter. The resulting isotonic solutionis then incorporated into ophthalmic delivery units, such as eye dropcontainers, which are suitable for ophthalmic administration. Two dropsof the composition is applied to a burn-damaged eye 4 times per day. Byuse of this method, the eye returns to normal health.

Example 7 Eye Ointment Composition and Treatment of Eye Disease usingSame

A sterile eye ointment composition is prepared by compounding 90 gramswhite petrolatum, 10 grams liquid petrolatum, and 0.5 grams lyophilizedAM powder. The mixture is pasteurized and packaged into individual tubecontainers of 2.0 g each.

To treat an eye disease using the composition, an aliquot ofapproximately 0.1 g is gently applied directly from the tube to theinner edge of the bottom eye lid. The ointment is applied 4 times perday. The patient progress is monitored every other week by anopthamologist. By use of this method, the eye disease improves.

Example 8 Treatment of Human Eye Disease using AM Preparation

An individual with burn damage to the eye is identified. A preparationof 1% AM, 0.5% collagen in DMEM is prepared. The individual is treated4× per day with 2 drops of the composition. By use of this method, theeye damage improves as compared to a non-treated burn damaged eye.

Example 9 Treatment of Human Skin Disease using AM Preparation

An individual with psoriasis is identified. The individual is treatedwith a 5% preparation of reconstituted AM, supplemented with 1 mg/mlpurified Smad7 derived from a commercial source. The formulation isdissolved in a lotion composition. The treatment is administered 2 timesper day. By use of this method, the psoriasis is alleviated ordisappears.

Example 10 Rectal Gel Composition Containing AM Extract

A rectal gel composition is prepared by combining 100 mg of commerciallyavailable HA and TSG-6 (purified) with 5 ml sterile AM extract preparedfrom frozen AM membrane material as described in Example 1. To thismixture is added 2.5 g of methylcelluose (1500 mPa), 100 mg ofmethylparapen, 5 g of glycerin and 95 mL of purified water. Theresulting gel mixture is then incorporated into rectal delivery units,such as syringes, which are suitable for rectal administration.

Example 11 Parenteral Composition

A parenteral composition for intramuscular administration is prepared bymixing 10 mg each of: HA, TSG-6, PTX-3, and TSP-1, each of which isobtained from a commercial source, with 100 mg of a water-soluble saltof a compound described herein is dissolved in DMSO and then mixed with10 mL of 0.9% sterile saline. The mixture is incorporated into a dosageunit form suitable for administration by injection.

Example 12 Treatment of a Human Tumor by Direct Injection of an AMParenteral Preparation

A patient having a subcutaneous tumor of approximately 2 cm in width isidentified. 10 grams of liquid AM extract is mixed with 10% PEG 300 inwater for 2 hours at 4° C. The mixture is filtered through a 0.20 μmfilter, and aliquoted to sterile glass vials which were closed withrubber stoppers and sealed with aluminum caps. The vials are stored at4° C. for up to 2 weeks prior to use. The patient is treated by directlyinjecting the 0.50 ml solution through the skin into the tumor site,dividing the administration volume into 4 separate regions of the tumormass (approximately 125 μl to each of the four sites). The compositionis administered every 48 hours. The tumor size is monitored weekly. Byuse of this method the tumor size decreases.

While preferred embodiments have been shown and described herein,numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein can be employed in practicing the invention. It isintended that the following claims define the scope of the invention andthat methods and structures within the scope of these claims and theirequivalents be covered thereby.

1-10. (canceled)
 11. A pharmaceutical composition comprising: pulverizedor ground amniotic membrane preparation prepared from placenta that isfrozen or previously frozen.
 12. The composition of claim 11, whereinthe composition is formulated as a non-solid dosage form.
 13. Thecomposition of claim 11, wherein the composition is formulated as asolution, drops, suspension, aerosol, paste, spray, ointment, oil,emulsion, cream, lotion, gel, a coated bandage, a patch, sticks, balms,shampoo, or combination thereof.
 14. The composition of claim 11,wherein the composition is formulated for ophthalmic administration. 15.The composition of claim 15, wherein the composition is formulated as anintraocular injection, subretinal injection, intravitreal injection,periocular administration, subconjuctival injection, retrobulbarinjection, intracameral injection, sub-Tenon's injection, implant,ophthalmic solution, ophthalmic suspension, ophthalmic ointment, ocularimplant, ocular insert, or intraocular solution.
 16. The composition ofclaim 11, wherein the composition is for topical administration.
 17. Thecomposition of claim 11, wherein the composition further comprises apenetration enhancer and an aqueous adjuvant.
 18. The composition ofclaim 11, wherein the composition is for systemic administration. 19.The composition of claim 11, wherein the composition is formulated as asolid dosage form.
 20. The composition of claim 11, wherein thecomposition is formulated as a solid dosage form, and wherein the soliddosage form is for inserting between an eye and an eyelid, or in aconjunctival sac.
 21. The composition of claim 11, wherein thecomposition is formulated as a solid oral dosage form.
 22. Thecomposition of claim 11, wherein the composition is formulated as acapsule or tablet.
 23. The composition of claim 11, wherein thecomposition is formulated for controlled release.
 24. The composition ofclaim 11, wherein the composition is formulated as an enteric coatedoral dosage form.
 25. The composition of claim 11, wherein the amnioticmembrane (M preparation comprises high molecular weight hyaluronan (HA)that is cross-linked by a covalent bond to the heavy chain ofinter-α-trypsin inhibitor (IαI), the high molecular weight HA having amolecular weight greater than 1000 kDa.
 26. The composition of claim 11,wherein the amniotic membrane AM preparation comprises tumor necrosisfactor-stimulated gene 6 (TSG-6), pentraxin (PTX-3), thrombospondin(TSP-1), and cross-linked high molecular weight hyaluronan (HA). 27.(canceled)
 28. (canceled)
 29. The composition of claim 11, wherein thecomposition is a pharmaceutical composition and further comprises apharmaceutically acceptable excipient.